EP2453024A2 - Genes and pathways differentially expressed in bipolar disorder and/or major depressive disorder - Google Patents

Genes and pathways differentially expressed in bipolar disorder and/or major depressive disorder Download PDF

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EP2453024A2
EP2453024A2 EP20110175854 EP11175854A EP2453024A2 EP 2453024 A2 EP2453024 A2 EP 2453024A2 EP 20110175854 EP20110175854 EP 20110175854 EP 11175854 A EP11175854 A EP 11175854A EP 2453024 A2 EP2453024 A2 EP 2453024A2
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protein
expression
disorder
gene
antagonist
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EP2453024A3 (en
EP2453024B1 (en
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Margherita Molnar
Huda Akil
William E. Bunney
Prabhakara V. Choudary
Simon J. Evans
Edward G. Jones
Jun Li
Juan F. Lopez
David M. Lyons
Richard M. Myers
Alan F. Schatzberg
Richard Stein
Robert C. Thompson
Hiroaki Tomita
Marquis P. Vawter
Stanley J. Watson
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Leland Stanford Junior University
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Leland Stanford Junior University
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Definitions

  • the present invention provides methods for diagnosis and treatment of mental illness, as well as methods for identifying compounds effective in treating mental illness.
  • the inventors of the present application have used DNA microarrays to study expression profiles of human post-mortem brains from patients diagnosed with BP or MDD.
  • the present invention relates to differential gene expression in the Anterior Cingulate (AnCg), Dorsolateral Prefrontal (DLPFC), and Cerebellar (CB) cortices, Hippocampus, Nucleus Accumbens and Amygdala regions of the brain, wherein the differential gene expression is associated with Bipolar Disorder (BPD) and Major Depressive Disorder (MDD).
  • genes in these regions are considered "unique" to a given disorder such as BP or MDD when differentially expressed in a particular mood disorder and not another (see, e.g., Tables 3, 4, 14-20). In other cases, where the genes are differentially expressed in both BP and MDD relative to healthy controls, the genes are considered to be involved in both disorders. Expression of the differentially expressed genes may be detected using any suitable methods of detection, e.g., microarrays, PCR or in situ hybridization. Gene expression may be detected in brain tissue, brain tissue samples, or other tissue samples ( e.g ., blood samples in the case of NCAM1).
  • the present invention relates to differential gene expression associated with G protein-coupled receptors (GPCR)s and downstream signaling pathways, mediated by cyclic adenosine monophosphate (cAMP) and phosphatidylinositol (PI), wherein the gene expression differentially occurs in the Anterior Cingulate (AnCg), Dorsolateral Prefrontal (DLPFC), and Cerebellar (CB) cortices, Hippocampus, Nucleus Accumbens and Amygdala regions of the brains of patients with Bipolar Disorder (BPD) and/or Major Depressive Disorder (MDD), relative to healthy controls (see, e.g., Tables 14-20).
  • GPCR G protein-coupled receptors
  • PI phosphatidylinositol
  • the present invention relates to differential gene expression associated with G protein-coupled receptors (GPCR)s and downstream signaling pathways, mediated by cyclic adenosine monophosphate (cAMP) and phosphatidylinositol (PI), wherein the gene expression differentially occurs in the Hippocampus, Nucleus Accumbens and Amygdala regions of the brains of patients with Bipolar Disorder (BPD) and/or Major Depressive Disorder (MDD), relative to healthy controls (see, e.g., Tables 18-20).
  • GPCR G protein-coupled receptors
  • cAMP cyclic adenosine monophosphate
  • PI phosphatidylinositol
  • the present invention also demonstrates differential expression of the FGF pathway in the frontal cortex of MDD subjects.
  • FGF-related genes such as FGF2 are dysregulated by antidepressant therapy, environmental complexity, and the correlation to anxiety-like behavior (see, e.g., Tables 1a, 1b, and 2, and Figures 1-7 and 22 ).
  • the FGF pathway is also related to neurogenesis, e.g., neural stem cell proliferation and differentiation, and the genes disclosed herein can be used for diagnosis and therapeutics related to neurogenesis.
  • Figure 23 shows the effects of postnatal FGF-2 adminstration on neurogenesis, emotionality and gene expression in adult rats.
  • the FGF injected animals exhibit significantly increased cell survival and proliteration in the dentate gyrus of the hippocampus. As adults, the animals show higher locomotor activity in a novel environment, an index of lower anxiety, and have better learning and memory.
  • the present invention also demonstrates that the genes of the glutamate/GABA signaling pathways are involved in MDD and BP (see Figure 24 and Table 8).
  • the present invention also demonstrates that mitrochondrial genes are involved in MDD andBP (see Table 10).
  • the present invention also demonstrates that 40 genes encoding growth factor family members and growth factor receptors are significantly differentially expressed in BP or MDD in the DLPFC or AnCg (see Tables 5, 6 and 7).
  • the present invention demonstrates that genes involved in G protein coupled receptors and their downstream signaling pathways , including cyclic AMP, phosphatidylinositol, and mitogen-activated protein kinase signaling pathways are dysregulated in BP and/or MDD (see Tables 6 and 9 and Figures 8-13 and 17-19 ).
  • the present invention provides for the first time a novel insertion/deletion polymporphism in the phosphoserine phosphatase-like gene (PSPHL) and demonstrates that a novel deletion polymorphism of PSPHL is related to susceptibility to bipolar disorder. Therefore, detection of this polymorphism is useful for diagnosis of BP, as well as for drug discovery assays for BP therapeutics.
  • the serine amino acid metabolic pathway, of which PSPHL is a member is a target for drug discovery for BP therapeutics.
  • the PSPHL gene was first cloned by Planitzer et al., Gene 210 297-306 (1998 ).
  • the accession number for a representative nucleic acid sequence is AJ0016112 and the accession number for a representative protein sequence is CAA04865.1. See Figures 14-16 .
  • the present invention demonstrates, for the first time, unique expression of the 24 nucleic acids listed in Table 3 in the brains of bipolar disorder subjects but not major depression subjects; the unique expression of the 24 nucleic acids listed in Table 4 in the brains of major depression subjects but not bipolar subjects, and the differential and/or unique expression of the nucleic acids listed in Tables 5-10 in the brains of patients suffering from bipolar disorder and major depression disorder, in comparison with normal control subjects.
  • the present invention identifies biochemical pathways involved in uniquely or differentially in mood disorders, where the proteins encoded by the nucleic acids listed in Table 3-10 are components of the biochemical pathways (e.g., the growth factor, e.g., FGF, signal transduction pathway, GPCR signal transduction pathways, mitochondrial pathways, and glutamate/GABA signaling pathways).
  • the invention demonstrates the unique expression of a PSPHL deletion polymorphism and it's associate with BP.
  • Genes and pathways that are uniquely or differentially expressed in MDD or BP are useful in diagnosing mood disorders and in assaying for therapeutics that can specifically treat MDD or BP, or can be used to treat both MDD and BP.
  • Differential expression by brain region similarly is a useful diagnostic and therapeutic tool, as certain mood disorders primarily affect certain brain regions. Each brain region plays a unique and critical role in the overall phenotype of any particular mood disorder.
  • the gene described herein unique to BP can also be uniquely expressed in schizophrenia, and so can be used for differential diagnosis with MDD.
  • This invention thus provides methods for determining whether a subject has or is predisposed for a mental disorder such as bipolar disorder or major depression disorder.
  • the invention also provides methods of providing a prognosis and for monitoring disease progression and treatment.
  • the present invention provides nucleic acid and protein targets for assays for drugs for the treatment of mental disorders such as bipolar disorder and major depression disorder.
  • the methods comprise the steps of: (i) obtaining a biological sample from a subject; (ii) contacting the sample with a reagent that selectively associates with a polynucleotide or polypeptide encoded by a nucleic acid that hybridizes under stringent conditions to a nucleotide sequence listed in Tables 3-10 and Figure 14 ; and (iii) detecting the level of reagent that selectively associates with the sample, thereby determining whether the subject has or is predisposed for a mental disorder.
  • the reagent is an antibody. In some embodiments, the reagent is a nucleic acid. In some embodiments, the reagent associates with a polynucleotide. In some embodiments, the reagent associates with a polypeptide. In some embodiments, the polynucleotide comprises a nucleotide sequence listed in Table 3-6. In some embodiment, the polypeptide comprises an amino acid sequence of a gene listed in Table 3-6. In some embodiments, the level of reagent that associates with the sample is different (i.e., higher or lower) from a level associated with humans without a mental disorder. In some embodiments, the biological sample is obtained from amniotic fluid. In some embodiments, the mental disorder is a mood disorder. In some embodiments, the mood disorder is selected from the group consisting of bipolar disorder and major depression disorder.
  • the invention also provides methods of identifying a compound for treatment of a mental disorder.
  • the methods comprises the steps of: (i) contacting the compound with a polypeptide, which is encoded by a polynucleotide that hybridizes under stringent conditions to a nucleic acid comprising a nucleotide sequence of Table 2 , 3, or 4; and (ii) determining the functional effect of the compound upon the polypeptide, thereby identifying a compound for treatment of a mental disorder.
  • the contacting step is performed in vitro.
  • the polypeptide comprises an amino acid sequence of a gene listed in Table 3-6.
  • the polypeptide is expressed in a cell or biological sample, and the cell or biological sample is contacted with the compound.
  • the mental disorder is a mood disorder or psychotic disorder.
  • the mood disorder is selected from the group consisting of bipolar disorder and major depression.
  • the psychotic disorder is schizophrenia.
  • the methods further comprise administering the compound to an animal and determining the effect on the animal, e.g., an invertebrate, a vertebrate, or a mammal. In some embodiments, the determining step comprises testing the animal's mental function.
  • the methods comprise the steps of (i) contacting the compound to a cell, the cell comprising a polynucleotide that hybridizes under stringent conditions to a nucleotide sequence of Table 3-6; and (ii) selecting a compound that modulates expression of the polynucleotide, thereby identifying a compound for treatment of a mental disorder.
  • the polynucleotide comprises a nucleotide sequence listed in Table 3-6.
  • the expression of the polynucleotide is enhanced.
  • the expression of the polynucleotide is decreased.
  • the methods further comprise administering the compound to an animal and determining the effect on the animal.
  • the determining step comprises testing the animal's mental function.
  • the mental disorder is a mood disorder or a psychotic disorder.
  • the mood disorder is selected from the group consisting of bipolar disorder and major depression.
  • the psychotic disorder is schizophrenia.
  • the invention also provides methods of treating a mental disorder in a subject.
  • the methods comprise the step of administering to the subject a therapeutically effective amount of a compound identified using the methods described above.
  • the mental disorder is a mood disorder or a psychotic disorder.
  • the mood disorder is selected from the group consisting of bipolar disorder and major depression.
  • the psychotic disorder is schizophrenia.
  • the compound is a small organic molecule, an antibody, an antisense molecule, or a peptide.
  • the invention also provides methods of treating mental illness in a subject, comprising the step of administering to the subject a therapeutically effective amount of a polypeptide, which is encoded by a polypeptide that hybridizes under stringent conditions to a nucleic acid of Table 3-6.
  • the polypeptide comprises an amino acid sequence encoded by a gene listed in Table 3-6.
  • the mental illness is a mood disorder or a psychotic disorder.
  • the psychotic disorder is schizophrenia.
  • the mood disorder is a bipolar disorder or major depression.
  • the invention also provides methods of treating mental illness in a subject, comprising the step of administering to the subject a therapeutically effective amount of a polypeptide, wherein the polypeptide hybridizes under stringent conditions to a nucleic acid of Table 3-6.
  • the mental illness is a mood disorder or a psychotic disorder.
  • the psychotic disorder is schizophrenia.
  • the mood disorder is a bipolar disorder or major depression.
  • Figure 1 Rodent FGFR2 ISH.
  • Figure 2 Figure 2 summarizes differential expression of FGF system transcripts in MDD cortex.
  • Figure 3 shows FGF dysregulation is attenuated by anti-depressant therapy.
  • Figure 4 shows that chronic fluoxetine treatment increases FGFR2 expression in rat forebrain.
  • Figure 5 shows environmental complexity: FGF2 and anxiety-like behavior.
  • Figure 6 shows FGF expression negatively correlates with anxiety-like behavior.
  • Figure 7 shows summarizes FGF and negative affect.
  • Figure 8 shows qRT-PCR validation of microarray data.
  • Figure 9 shows G-protein coupled receptor (GPCR) and ligands dysregulated in anterior cingulated cortex of BP subjects.
  • GPCR G-protein coupled receptor
  • Figure 10 shows gene category over-representation analysis of the three downstream GPCR signaling pathways.
  • Figure 11 shows phosphatidylinositol metabolism in BP disorder.
  • Figure 12 shows mitogen activated protein kinase signaling in BP disorder.
  • Figure 13 shows cAMP signaling pathway in BP subjects
  • Figure 14 shows genomic structure of the PSPHL gene and the deletion polymorphism of PSPHL that is related to BP susceptibility.
  • Figure 15 shows the predicted amino acid sequences for PSPH, PSPHL-A, and PSPHL-B.
  • Figure 16 shows the serine amino acid metabolic pathway.
  • Figure 17 shows where each exon begins and ends in the PSPHL mRNA and provides primers to detect insertion/deletion polymorphisms in the PSPHL locus.
  • Figure 18 shows a gel image for PSPHL insertion/deletion alleles.
  • Figure 19 shows the cAMP signaling pathway in the limbic system for BP.
  • Figure 20 shows the PI signaling pathwayi in the limbic system for MDD.
  • Figure 21 shows MAPK signaling pathway in the limbic system for MDD.
  • Figure 22 shows the effects of environmental complexity on differences in anxiety behavior and FGF2 gene expression.
  • Figure 23 shows the effects of postnatal FGF2 administration on neurogenesis, emotionality and gene expression in adult rats.
  • Figure 24 shows GABA/glutamate signaling pathways in BP and MDD.
  • Figure 25 shows NCAM SNPs and splice variants involved in mood disorders such as bipolar disorder.
  • Figure 26 The figure summarizes differential expressed genes regulating cAMP- (A, B) and phosphatidylinositol- (C, D) signaling pathways in the brain of BPD (A, C) and MDD (B, D).
  • GNAI1 G protein alpha inhibiting activity 1
  • RGS20 Regulator of G-protein signaling 20
  • PDE1A Phosphodiesterase 1A
  • PDE8A Phosphodiesterase 8A
  • PKIA Protein kinase A inhibitor alpha
  • CDK5 Cyclin-dependent kinase 5
  • PPP1CA Protein phosphatase 1, catalytic alpha
  • PPP1R3C Protein phosphatase 1, regulatory 3C
  • INPP5A Inositol polyphosphate-5-phosphatase A
  • INPP5F Inositol polyphosphate-5-phosphatase F
  • ITPKB Inositol 1,4,5-trisphosphate 3-kinase B
  • INPP1 I
  • FIG. 27 In situ hybridization images of GPR37 mRNA in representative BPD, MDD and control subjects GPR37 mRNA is preferentially expressed in subcortical white matter. Among 6 layers of cortical gray matter, GPR37 expression in deeper layer (V-VI) is relatively higher than superficial layer (I-III). Expression levels in GPR37 is increased in the BPD subjects, and decreased in MDD subjects in subcortical white matter in anterior cingulate cortex tissue, compared to control subjects.
  • WM Subcortical white matter
  • BPD Bipolar disorder
  • MDD Major depressive disorder.
  • Figures 28 and 29 In situ hybridization for LRPPRC (leucine-rich PPR-motif containing) mRNA in three brain regions.
  • A LRPPRC expression in BPD and control representative images.
  • NCAM1 i.e., neural cell adhesion molecule 1 genomic organization and location of four polymorphic sites. The gene spans 214 kb, but does not contain any exonic SNPs. The arrows indicate the location of the four polymorphisms and the five exons used in this exploratory analysis.
  • Figure 31 Significant alterations of NCAM1 exon splice variant levels are shown by genotype and diagnosis by genotype.
  • Table 1a Table 1a lists subject data for cohort A.
  • Table 1b Table 1b lists subject data for cohort B.
  • Table 2 shows microarray data for all FGF transcripts detected in either DLPFC or AnCg and summary data for confirmation studies.
  • Table 3 Table 3 lists genes uniquely expressed in BP subjects.
  • Table 4 Table 4 lists genes uniquely expressed in MDD subjects.
  • Table 5 Table 5 lists growth factor pathway genes expressed in MDD and BP subjects.
  • Table 6 Table 6 lists GPCR pathway genes expressed in MDD and BP subjects.
  • Table 7 lists growth factor pathway genes expressed in MDD and BP subjects.
  • Table 8 lists GABA and glutamate signaling pathway genes expressed in MDD and BP subjects.
  • Table 9 lists GPCR pathway genes expressed in MDD and BP subjects.
  • Table 10 Table 10 lists mitochondrial genes expressed in MDD and BP subjects.
  • Table 11 Table 11 lists genes expressed in MDD, BP, and schizophrenia subjects.
  • Table 12 Table 12 lists genes expressed in MDD, BP, and schizophrenia subjects.
  • Table 13 lists GPCR pathway genes expressed in MDD and BP.
  • Table 14 GPCRs and related signaling genes dysregulated in anterior cingulate cortex.
  • Table 15 GPCRs and related signaling genes dysregulated in dorsolateral prefrontal cortex.
  • Table 16 GPCRs and related signaling genes dysregulated in cerebellar cortex.
  • Table 17 Quantitative RT-PCR data. Fold changes in microarray and qRT-PCR analyses for representative ligand peptides, GPCRs, G protein regulator (NPY, SST, GPR37, GPRC5B, RGS20), which were dysregulated in BPD/MDD compared to the control group. N.S., No significant change; *. p ⁇ 0.05; **, p ⁇ 0.01.
  • Table 18 GPCRs and related signaling genes dysregulated in amygdala, hippocampus, nucleus accumbens of BPD.
  • Table 19 GPCRs and related signaling genes dysregulated in amygdala, hippocampus, nucleus accumbens of MDD.
  • Table 20 shows the genes that were differentially expressed in BPD or MDD by > 1.2 fold change and were down-regulated in agonal factor control comparisons by ⁇ 1.0. The opposite genes are also shown, where there was a decrease in mood disorder by ⁇ -1.2 fold change, and the agonal factor control comparison showed an increase > 1.0 fold change. These genes were found in 4 major classifications listed: mitochondria, chaperone, apoptosis, and proteasome.
  • Table 21 Real time Q-PCR validation results for selected mitochondrial related candidate genes for mood disorders in two cortical regions. These genes are nuclear-encoded. Significant by Q-PCR p ⁇ 0.05 one-tailed t-test. The Q-PCR t-test MDD, BPD, and control groups used subjects with no agonal factors and pH > 6.8 similar to microarray analysis #3 groups MDD-High, BPD-High, and Control-High.
  • mtDNA Mitochondrial DNA (mtDNA) encoded genes were analyzed by real time Q-PCR for differential expression in BPD and MDD compared to controls. Nuclear encoded genes in BPD and MDD subjects appeared to generally be increased while several mtDNA genes showed a significant decrease by Q-PCR in mood disorders.
  • Table 23 Primers for each DNA segment and possible combination of splice variants (a, b, c, SEC and VASE), as well as, for SNP 9 and an exon outside of the splice sites for measuring total NCAM1. The numbering is shown in Figure 26 according to accession M22094. *1 - Only the forward primer could be designed because exon a is 14 bps. 2 - Exon 3 is before the variable exons and only the forward primer was needed to PCR outside the exons. 3 - Exon 8 is after the variable exons and only the reverse primer was needed to PCR outside the exons.
  • Table 24 Genotypic Association Results. The odds ratio, chi-square (chi2) and p-values where all calculated using the DeFinetti program Tests for Deviation from HWE and Tests for Association (C.I.: 95% confidence interval).
  • Table 25 SNP 9 and SNP b haplotype frequency, odds ratio and p-values. P-values were calculated from the Chi-squared values derived from the EHplus program.
  • Table 26 Genotypic and Allelic Distributions for Controls, Bipolar Disorder and Schizophrenia. Fisher's exact p-values are shown for allelic distribution between case-controls.
  • Table 27 Genotype x Splice Variant Differences x Diagnosis (p-values). For each SNP genotype and splice variant the splice variant amounts were evaluated by t-test based on diagnosis and the significant p-values were reported.
  • Tables 28 Genes upregulated (28.1) and downregulated (28.2) by Lithium in monkey brains.
  • Table 29 Values of V-ATPase Subunits differential expression in Non-human primate model of depression.
  • CRS chronic unpredictable stress
  • All fluoxetine, desipramine, and bupropion. Controls were administered water (H2O treated).
  • a “mental disorder” or “mental illness” or “mental disease” or “psychiatric or neuropsychiatric disease or illness or disorder” refers to mood disorders (e.g., major depression, mania, and bipolar disorders), psychotic disorders (e.g., schizophrenia, schizoaffective disorder, schizophreniform disorder, delusional disorder, brief psychotic disorder, and shared psychotic disorder), personality disorders, anxiety disorders (e.g., obsessive-compulsive disorder) as well as other mental disorders such as substance -related disorders, childhood disorders, dementia, autistic disorder, adjustment disorder, delirium, multi-infarct dementia, and Tourette's disorder as described in Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, (DSM IV ). Typically, such disorders have a complex genetic and/or a biochemical component.
  • a "mood disorder” refers to disruption of feeling tone or emotional state experienced by an individual for an extensive period of time.
  • Mood disorders include major depression disorder (i.e., unipolar disorder), mania, dysphoria, bipolar disorder, dysthymia, cyclothymia and many others. See, e.g., Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, (DSM IV).
  • Major depression disorder refers to a mood disorder involving any of the following symptoms: persistent sad, anxious, or “empty” mood; feelings of hopelessness or pessimism; feelings of guilt, worthlessness, or helplessness; loss of interest or pleasure in hobbies and activities that were once enjoyed, including sex; decreased energy, fatigue, being “slowed down”; difficulty concentrating, remembering, or making decisions; insomnia, early-morning awakening, or oversleeping; appetite and/or weight loss or overeating and weight gain; thoughts of death or suicide or suicide attempts; restlessness or irritability; or persistent physical symptoms that do not respond to treatment, such as headaches, digestive disorders, and chronic pain.
  • Various subtypes of depression are described in, e.g., DSM IV.
  • Bipolar disorder is a mood disorder characterized by alternating periods of extreme moods. A person with bipolar disorder experiences cycling of moods that usually swing from being overly elated or irritable (mania) to sad and hopeless (depression) and then back again, with periods of normal mood in between. Diagnosis of bipolar disorder is described in, e.g., DSM IV. Bipolar disorders include bipolar disorder I (mania with or without major depression) and bipolar disorder II (hypomania with major depression), see, e.g., DSM IV.
  • a psychotic disorder refers to a condition that affects the mind, resulting in at least some loss of contact with reality. Symptoms of a psychotic disorder include, e.g., hallucinations, changed behavior that is not based on reality, delusions and the like. See, e.g., DSM IV. Schizophrenia, schizoaffective disorder, schizophreniform disorder, delusional disorder, brief psychotic disorder, substance-induced psychotic disorder, and shared psychotic disorder are examples of psychotic disorders.
  • “Schizophrenia” refers to a psychotic disorder involving a withdrawal from reality by an individual. Symptoms comprise for at least a part of a month two or more of the following symptoms: delusions (only one symptom is required if a delusion is playful, such as being abducted in a space ship from the sun); hallucinations (only one symptom is required if hallucinations are of at least two voices talking to one another or of a voice that keeps up a running commentary on the patient's thoughts or actions); disorganized speech (e.g., frequent derailment or incoherence); grossly disorganized or catatonic behavior; or negative symptoms, i.e., affective flattening, alogia, or avolition.
  • delusions only one symptom is required if a delusion is playful, such as being abducted in a space ship from the sun
  • hallucinations only one symptom is required if hallucinations are of at least two voices talking to one another or of a
  • Schizophrenia encompasses disorders such as, e.g., schizoaffective disorders. Diagnosis of schizophrenia is described in, e.g., DSM IV. Types of schizophrenia include, e.g., paranoid, disorganized, catatonic, undifferentiated, and residual.
  • an “antidepressant” refers to an agents typically used to treat clinical depression.
  • Antidepressants includes compounds of different classes including, for example, specific serotonin reuptake inhibitors (e.g., fluoxetine), tricyclic antidepressants (e.g., desipramine), and dopamine reuptake inhibitors (e.g, bupropion).
  • specific serotonin reuptake inhibitors e.g., fluoxetine
  • tricyclic antidepressants e.g., desipramine
  • dopamine reuptake inhibitors e.g, bupropion
  • antidepressants of different classes exert their therapeutic effects via different biochemical pathways. Often these biochemical pathways overlap or intersect. Additonal diseases or disorders often treated with antidepressants include, chronic pain, anxiety disorders, and hot flashes.
  • An "agonist” refers to an agent that binds to a polypeptide or polynucleotide of the invention, stimulates, increases, activates, facilitates, enhances activation, sensitizes or up regulates the activity or expression of a polypeptide or polynucleotide of the invention.
  • an “antagonist” refers to an agent that inhibits expression of a polypeptide or polynucleotide of the invention or binds to, partially or totally blocks stimulation, decreases, prevents, delays activation, inactivates, desensitizes, or down regulates the activity of a polypeptide or polynucleotide of the invention.
  • Inhibitors “Inhibitors,” “activators,” and “modulators” of expression or of activity are used to refer to inhibitory, activating, or modulating molecules, respectively, identified using in vitro and in vivo assays for expression or activity, e.g., ligands, agonists, antagonists, and their homologs and mimetics.
  • modulator includes inhibitors and activators.
  • Inhibitors are agents that, e.g., inhibit expression of a polypeptide or polynucleotide of the invention or bind to, partially or totally block stimulation or enzymatic activity, decrease, prevent, delay activation, inactivate, desensitize, or down regulate the activity of a polypeptide or polynucleotide of the invention, e.g., antagonists.
  • Activators are agents that, e.g., induce or activate the expression of a polypeptide or polynucleotide of the invention or bind to, stimulate, increase, open, activate, facilitate, enhance activation or enzymatic activity, sensitize or up regulate the activity of a polypeptide or polynucleotide of the invention, e.g., agonists.
  • Modulators include naturally occurring and synthetic ligands, antagonists, agonists, small chemical molecules and the like.
  • Assays to identify inhibitors and activators include, e.g., applying putative modulator compounds to cells, in the presence or absence of a polypeptide or polynucleotide of the invention and then determining the functional effects on a polypeptide or polynucleotide of the invention activity.
  • Samples or assays comprising a polypeptide or polynucleotide of the invention that are treated with a potential activator, inhibitor, or modulator are compared to control samples without the inhibitor, activator, or modulator to examine the extent of effect. Control samples (untreated with modulators) are assigned a relative activity value of 100%.
  • Inhibition is achieved when the activity value of a polypeptide or polynucleotide of the invention relative to the control is about 80%, optionally 50% or 25-1%.
  • Activation is achieved when the activity value of a polypeptide or polynucleotide of the invention relative to the control is 110%, optionally 150%, optionally 200-500%, or 1000-3000% higher.
  • test compound or “drug candidate” or “modulator” or grammatical equivalents as used herein describes any molecule, either naturally occurring or synthetic, e.g., protein, oligopeptide (e.g., from about 5 to about 25 amino acids in length, preferably from about 10 to 20 or 12 to 18 amino acids in length, preferably 12, 15, or 18 amino acids in length), small organic molecule, polysaccharide, lipid, fatty acid, polynucleotide, RNAi, oligonucleotide, etc.
  • the test compound can be in the form of a library of test compounds, such as a combinatorial or randomized library that provides a sufficient range of diversity.
  • Test compounds are optionally linked to a fusion partner, e.g., targeting compounds, rescue compounds, dimerization compounds, stabilizing compounds, addressable compounds, and other functional moieties.
  • a fusion partner e.g., targeting compounds, rescue compounds, dimerization compounds, stabilizing compounds, addressable compounds, and other functional moieties.
  • new chemical entities with useful properties are generated by identifying a test compound (called a "lead compound") with some desirable property or activity, e.g., inhibiting activity, creating variants of the lead compound, and evaluating the property and activity of those variant compounds.
  • HTS high throughput screening
  • a "small organic molecule” refers to an organic molecule, either naturally occurring or synthetic, that has a molecular weight of more than about 50 Daltons and less than about 2500 Daltons, preferably less than about 2000 Daltons, preferably between about 100 to about 1000 Daltons, more preferably between about 200 to about 500 Daltons.
  • siRNA refers to a nucleic acid that forms a double stranded RNA, which double stranded RNA has the ability to reduce or inhibit expression of a gene or target gene when the siRNA expressed in the same cell as the gene or target gene.
  • siRNA or “RNAi” thus refers to the double stranded RNA formed by the complementary strands.
  • the complementary portions of the siRNA that hybridize to form the double stranded molecule typically have substantial or complete identity.
  • an siRNA refers to a nucleic acid that has substantial or complete identity to a target gene and forms a double stranded siRNA.
  • the siRNA is at least about 15-50 nucleotides in length (e.g., each complementary sequence of the double stranded siRNA is 15-50 nucleotides in length, and the double stranded siRNA is about 15-50 base pairs in length, preferable about preferably about 20-30 base nucleotides, preferably about 20-25 or about 24-29 nucleotides in length, e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.
  • Determining the functional effect refers to assaying for a compound that increases or decreases a parameter that is indirectly or directly under the influence of a polynucleotide or polypeptide of the invention (such as a polynucleotide of Table 3-6 or a polypeptide encoded by a gene of Table 3-6), e.g., measuring physical and chemical or phenotypic effects.
  • a polynucleotide or polypeptide of the invention such as a polynucleotide of Table 3-6 or a polypeptide encoded by a gene of Table 3-6
  • Such functional effects can be measured by any means known to those skilled in the art, e.g., changes in spectroscopic (e.g., fluorescence, absorbance, refractive index), hydrodynamic (e.g., shape), chromatographic, or solubility properties for the protein; measuring inducible markers or transcriptional activation of the protein; measuring binding activity or binding assays, e.g.
  • RNA stability e.g., G-protein binding; GPCR phosphorylation or dephosphorylation; signal transduction, e.g., receptor-ligand interactions, second messenger concentrations (e.g., cAMP, IP3, or intracellular Ca 2+ ); identification of downstream or reporter gene expression (CAT, luciferase, ⁇ -gal, GFP and the like), e.g., via chemiluminescence, fluorescence, colorimetric reactions, antibody binding, inducible markers, and ligand binding assays.
  • CAT reporter gene expression
  • Samples or assays comprising a nucleic acid or protein disclosed herein that are treated with a potential activator, inhibitor, or modulator are compared to control samples without the inhibitor, activator, or modulator to examine the extent of inhibition.
  • Control samples (untreated with inhibitors) are assigned a relative protein activity value of 100%. Inhibition is achieved when the activity value relative to the control is about 80%, preferably 50%, more preferably 25-0%.
  • Activation is achieved when the activity value relative to the control (untreated with activators) is 110%, more preferably 150%, more preferably 200-500% (i.e., two to five fold higher relative to the control), more preferably 1000-3000% higher.
  • Bio sample includes sections of tissues such as biopsy and autopsy samples, and frozen sections taken for histologic purposes. Such samples include blood, sputum, tissue, lysed cells, brain biopsy, cultured cells, e.g., primary cultures, explants, and transformed cells, stool, urine, etc.
  • a biological sample is typically obtained from a eukaryotic organism, most preferably a mammal such as a primate, e.g., chimpanzee or human; cow; dog; cat; a rodent, e.g., guinea pig, rat, mouse; rabbit; or a bird; reptile; or fish.
  • Antibody refers to a polypeptide substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof which specifically bind and recognize an analyte (antigen).
  • the recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes.
  • Light chains are classified as either kappa or lambda.
  • Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
  • An exemplary immunoglobulin (antibody) structural unit comprises a tetramer.
  • Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light” (about 25 kD) and one "heavy” chain (about 50-70 kD).
  • the N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • the terms variable light chain (V L ) and variable heavy chain (V H ) refer to these light and heavy chains respectively.
  • Antibodies exist, e.g., as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases.
  • pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)' 2 , a dimer of Fab which itself is a light chain joined to V H -C H 1 by a disulfide bond.
  • the F(ab)' 2 may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)' 2 dimer into an Fab' monomer.
  • the Fab' monomer is essentially an Fab with part of the hinge region (see, Paul (Ed.) Fundamental Immunology, Third Edition, Raven Press, NY (1993 )). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by utilizing recombinant DNA methodology. Thus, the term antibody, as used herein, also includes antibody fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv).
  • peptidomimetic and “mimetic” refer to a synthetic chemical compound that has substantially the same structural and functional characteristics of the polynucleotides, polypeptides, antagonists or agonists of the invention.
  • Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties analogous to those of the template peptide. These types of non-peptide compound are termed “peptide mimetics” or “peptidomimetics” ( Fauchere, Adv. Drug Res. 15:29 (1986 ); Veber and Freidinger TINS p. 392 (1985 ); and Evans et al., J. Med. Chem. 30:1229 (1987 ), which are incorporated herein by reference).
  • Peptide mimetics that are structurally similar to therapeutically useful peptides may be used to produce an equivalent or enhanced therapeutic or prophylactic effect.
  • the mimetic can be either entirely composed of synthetic, non-natural analogues of amino acids, or, is a chimeric molecule of partly natural peptide amino acids and partly non-natural analogs of amino acids.
  • the mimetic can also incorporate any amount of natural amino acid conservative substitutions as long as such substitutions also do not substantially alter the mimetic's structure and/or activity.
  • a mimetic composition is within the scope of the invention if it is capable of carrying out the binding or enzymatic activities of a polypeptide or polynucleotide of the invention or inhibiting or increasing the enzymatic activity or expression of a polypeptide or polynucleotide of the invention.
  • gene means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons).
  • nucleic acid or protein when applied to a nucleic acid or protein, denotes that the nucleic acid or protein is essentially free of other cellular components with which it is associated in the natural state. It is preferably in a homogeneous state although it can be in either a dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein that is the predominant species present in a preparation is substantially purified. In particular, an isolated gene is separated from open reading frames that flank the gene and encode a protein other than the gene of interest. The term “purified” denotes that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. Particularly, it means that the nucleic acid or protein is at least 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure.
  • nucleic acid or “polynucleotide” refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated.
  • conservatively modified variants thereof e.g., degenerate codon substitutions
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues ( Batzer et al., Nucleic Acid Res. 19:5081 (1991 ); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985 ); and Cassol et al. (1992); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994 )).
  • the term nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene.
  • polypeptide polypeptide
  • peptide protein
  • proteins proteins
  • amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.
  • the terms encompass amino acid chains of any length, including full-length proteins (i.e., antigens), wherein the amino acid residues are linked by covalent peptide bonds.
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, ⁇ -carboxyglutamate, and O-phosphoserine.
  • Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
  • Amino acids may be referred to herein by either the commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
  • Constantly modified variants applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, “conservatively modified variants” refers to those nucleic acids that encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide.
  • nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein that encodes a polypeptide also describes every possible silent variation of the nucleic acid.
  • each codon in a nucleic acid except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan
  • TGG which is ordinarily the only codon for tryptophan
  • amino acid sequences one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.
  • Percentage of sequence identity is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
  • nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity over a specified region), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Such sequences are then said to be “substantially identical.” This definition also refers to the complement of a test sequence. Optionally, the identity exists over a region that is at least about 50 nucleotides in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides in length.
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated.
  • sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
  • a “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (1970) Adv. Appl. Math. 2:482c , by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol.
  • BLAST and BLAST 2.0 algorithms are described in Altschul et al. (1977) Nuc. Acids Res. 25:3389-3402 , and Altschul et al. (1990) J. Mol. Biol. 215:403-410 , respectively.
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.
  • This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra ).
  • initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them.
  • the word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always ⁇ 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787 ).
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.
  • nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below.
  • a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions.
  • Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below.
  • Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.
  • stringent hybridization conditions refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acid, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays” (1993 ). Generally, stringent conditions are selected to be about 5-10° C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength pH.
  • T m thermal melting point
  • the T m is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at T m , 50% of the probes are occupied at equilibrium).
  • Stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C for long probes (e.g., greater than 50 nucleotides).
  • Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
  • a positive signal is at least two times background, optionally 10 times background hybridization.
  • Exemplary stringent hybridization conditions can be as following: 50% formamide, 5X SSC, and 1% SDS, incubating at 42°C, or 5X SSC, 1% SDS, incubating at 65°C, with wash in 0.2X SSC, and 0.1% SDS at 65°C. Such washes can be performed for 5, 15, 30, 60, 120, or more minutes. Nucleic acids that hybridize to the genes listed in Tables 3-10 and Figure 14 are encompassed by the invention.
  • Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides that they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cases, the nucleic acids typically hybridize under moderately stringent hybridization conditions.
  • Exemplary "moderately stringent hybridization conditions” include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37°C, and a wash in 1X SSC at 45°C. Such washes can be performed for 5, 15, 30, 60, 120, or more minutes. A positive hybridization is at least twice background. Those of ordinary skill will readily recognize that alternative hybridization and wash conditions can be utilized to provide conditions of similar stringency.
  • a temperature of about 36°C is typical for low stringency amplification, although annealing temperatures may vary between about 32°C and 48°C depending on primer length.
  • a temperature of about 62°C is typical, although high stringency annealing temperatures can range from about 50°C to about 65°C, depending on the primer length and specificity.
  • Typical cycle conditions for both high and low stringency amplifications include a denaturation phase of 90°C - 95°C for 30 sec - 2 min., an annealing phase lasting 30 sec. - 2 min., and an extension phase of about 72°C for 1 - 2 min. Protocols and guidelines for low and high stringency amplification reactions are provided, e.g., in Innis et al., PCR Protocols, A Guide to Methods and Applications (1990 ).
  • a nucleic acid sequence encoding refers to a nucleic acid that contains sequence information for a structural RNA such as rRNA, a tRNA, or the primary amino acid sequence of a specific protein or peptide, or a binding site for a trans-acting regulatory agent. This phrase specifically encompasses degenerate codons (i.e., different codons which encode a single amino acid) of the native sequence or sequences which may be introduced to conform with codon preference in a specific host cell.
  • recombinant when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified.
  • recombinant cells express genes that are not found within the native (nonrecombinant) form of the cell or express native genes that are otherwise abnormally expressed, under-expressed or not expressed at all.
  • heterologous when used with reference to portions of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature.
  • the nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source.
  • a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).
  • an "expression vector” is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a host cell.
  • the expression vector can be part of a plasmid, virus, or nucleic acid fragment.
  • the expression vector includes a nucleic acid to be transcribed operably linked to a promoter.
  • the specified antibodies bind to a particular protein and do not bind in a significant amount to other proteins present in the sample. Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein.
  • antibodies raised against a protein having an amino acid sequence encoded by any of the polynucleotides of the invention can be selected to obtain antibodies specifically immunoreactive with that protein and not with other proteins, except for polymorphic variants.
  • a variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein.
  • solid-phase ELISA immunoassays, Western blots, or immunohistochemistry are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See, Harlow and Lane Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, NY (1988 ) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.
  • a specific or selective reaction will be at least twice the background signal or noise and more typically more than 10 to 100 times background.
  • One who is "predisposed for a mental disorder” as used herein means a person who has an inclination or a higher likelihood of developing a mental disorder when compared to an average person in the general population.
  • the present invention therefore demonstrates the altered expression (either higher or lower expression as indicated herein) and in some cases unique differential expression of the genes of Tables 3-10 at the mRNA level in selected brain regions of patients diagnosed with mood disorders, as well as the PSPHL gene (see, e.g., Figure 14 ) (e.g., bipolar disorder and major depression disorder) in comparison with normal individuals.
  • This invention thus provides methods for diagnosis of mental disorders such as mood disorders (e.g., bipolar disorder, major depression, and the like) and other mental disorders having a genetic component by detecting the level of a transcript or translation product of the genes listed in Tables 3-10 and Figure 14 as well as their corresponding biochemical pathways.
  • the present invention relates to a novel insertion-deletion polymorphism of phosphoserine phosphatase-like gene, and the association between deletion allele of PSPHL and susceptibility to bipolar disorder (BPD).
  • BPD bipolar disorder
  • the invention therefore provides the first evidence linking a genetic variant of the PSPHL gene to bipolar disorder.
  • the finding will facilitate characterization of the physiological and pathological function of the gene relevant to bipolar disorder, and provides novel and significant use of this gene and its variants for diagnosis, treatment and prevention of bipolar disorder.
  • the invention further provides methods of identifying a compound useful for the treatment of such disorders by selecting compounds that modulates the functional effect of the translation products or the expression of the transcripts described herein.
  • the invention also provides for methods of treating patients with such mental disorders, e.g., by administering the compounds of the invention or by gene therapy.
  • genes and the polypeptides that they encode are useful for facilitating the design and development of various molecular diagnostic tools such as GeneChipsTM containing probe sets specific for all or selected mental disorders, including but not limited to mood disorders, and as an ante-and/or post-natal diagnostic tool for screening newborns in concert with genetic counseling.
  • Other diagnostic applications include evaluation of disease susceptibility, prognosis, and monitoring of disease or treatment process, as well as providing individualized medicine via predictive drug profiling systems, e.g., by correlating specific genomic motifs with the clinical response of a patient to individual drugs.
  • the present invention is useful for multiplex SNP and haplotype profiling, including but not limited to the identification of therapeutic, diagnostic, and pharmacogenetic targets at the gene, mRNA, protein, and pathway level.
  • Profiling of splice variants and deletions is also useful for diagnostic and therapeutic applications.
  • genes and the polypeptides that they encode, described herein, are also useful as drug targets for the development of therapeutic drugs for the treatment or prevention of mental disorders, including but not limited to mood disorders.
  • Antidepressants belong to different classes, e.g., desipramine, bupropion, and fluoxetine are in general equally effective for the treatment of clinical depression, but act by different mechanisms. The similar effectiveness of the drugs for treatment of mood disorders suggests that they act through a presently unidentified common pathway. Animal models of depression, including treatment of animals with known therapeutics such as SSRIs, can be used to examine the mode of action of the genes of the invention. Lithium is drug of choice for treating BP.
  • Mental disorders have a high co-morbidity with other neurological disorders, such as Parkinson's disease or Alzheimer's. Therefore, the present invention can be used for diagnosis and treatment of patients with multiple disease states that include a mental disorder such as a mood disorder.
  • mood disorders include BP, MDD, and other disorders such as psychotic-depression, depression and anxiety features, melancholic depression, chronic depression, BPI and BPII.
  • polynucleotides of the invention will be isolated and cloned using recombinant methods.
  • Such polynucleotides include, e.g., those listed in Tables 3-10 and Figure 14 , which can be used for, e.g., protein expression or during the generation of variants, derivatives, expression cassettes, to monitor gene expression, for the isolation or detection of sequences of the invention in different species, for diagnostic purposes in a patient, e.g., to detect mutations or to detect expression levels of nucleic acids or polypeptides of the invention.
  • the sequences of the invention are operably linked to a heterologous promoter.
  • the nucleic acids of the invention are from any mammal, including, in particular, e.g., a human, a mouse, a rat, a primate, etc.
  • nucleic acids sizes are given in either kilobases (kb) or base pairs (bp). These are estimates derived from agarose or acrylamide gel electrophoresis, from sequenced nucleic acids, or from published DNA sequences.
  • kb kilobases
  • bp base pairs
  • proteins sizes are given in kilodaltons (kDa) or amino acid residue numbers. Proteins sizes are estimated from gel electrophoresis, from sequenced proteins, from derived amino acid sequences, or from published protein sequences.
  • Oligonucleotides that are not commercially available can be chemically synthesized according to the solid phase phosphoramidite triester method first described by Beaucage & Caruthers, Tetrahedron Letts. 22:1859-1862 (1981 ), using an automated synthesizer, as described in Van Devanter et. al., Nucleic Acids Res. 12:6159-6168 (1984 ). Purification of oligonucleotides is by either native acrylamide gel electrophoresis or by anion-exchange HPLC as described in Pearson & Reanier, J. Chrom. 255:137-149 (1983 ).
  • the sequence of the cloned genes and synthetic oligonucleotides can be verified after cloning using, e.g., the chain termination method for sequencing double-stranded templates of Wallace et al., Gene 16:21-26 (1981 ).
  • the nucleic acids encoding the subject proteins are cloned from DNA sequence libraries that are made to encode cDNA or genomic DNA.
  • the particular sequences can be located by hybridizing with an oligonucleotide probe, the sequence of which can be derived from the sequences of the genes listed in Tables 3-10 and Figure 14 , which provide a reference for PCR primers and defines suitable regions for isolating specific probes.
  • the sequence is cloned into an expression library
  • the expressed recombinant protein can be detected immunologically with antisera or purified antibodies made against a polypeptide comprising an amino acid sequence encoded by a gene listed in Table 1-8.
  • Brain cells are an example of suitable cells to isolate RNA and cDNA sequences of the invention.
  • a source that is rich in mRNA The mRNA can then be made into cDNA, ligated into a recombinant vector, and transfected into a recombinant host for propagation, screening and cloning.
  • the DNA is extracted from a suitable tissue and either mechanically sheared or enzymatically digested to yield fragments of preferably about 5-100 kb. The fragments are then separated by gradient centrifugation from undesired sizes and are constructed in bacteriophage lambda vectors. These vectors and phage are packaged in vitro, and the recombinant phages are analyzed by plaque hybridization. Colony hybridization is carried out as generally described in Grunstein et al., Proc. Natl. Acad. Sci. USA., 72:3961-3965 (1975 ).
  • An alternative method combines the use of synthetic oligonucleotide primers with polymerase extension on an mRNA or DNA template.
  • Suitable primers can be designed from specific sequences of the invention.
  • This polymerase chain reaction (PCR) method amplifies the nucleic acids encoding the protein of interest directly from mRNA, cDNA, genomic libraries or cDNA libraries. Restriction endonuclease sites can be incorporated into the primers.
  • Polymerase chain reaction or other in vitro amplification methods may also be useful, for example, to clone nucleic acids encoding specific proteins and express said proteins, to synthesize nucleic acids that will be used as probes for detecting the presence of mRNA encoding a polypeptide of the invention in physiological samples, for nucleic acid sequencing, or for other purposes (see, U.S. Patent Nos. 4,683,195 and 4,683,202 ).
  • Genes amplified by a PCR reaction can be purified from agarose gels and cloned into an appropriate vector.
  • PCR Protocols A Guide to Methods and Applications, Academic Press, San Diego (1990 ).
  • Synthetic oligonucleotides can be used to construct genes. This is done using a series of overlapping oligonucleotides, usually 40-120 bp in length, representing both the sense and anti-sense strands of the gene. These DNA fragments are then annealed, ligated and cloned.
  • a gene encoding a polypeptide of the invention can be cloned using intermediate vectors before transformation into mammalian cells for expression.
  • These intermediate vectors are typically prokaryote vectors or shuttle vectors.
  • the proteins can be expressed in either prokaryotes, using standard methods well known to those of skill in the art, or eukaryotes as described infra.
  • Either naturally occurring or recombinant polypeptides of the invention can be purified for use in functional assays.
  • Naturally occurring polypeptides e.g., polypeptides encoded by genes listed in Tables 3-10 and Figure 14
  • Recombinant polypeptides can be purified from any suitable expression system.
  • polypeptides of the invention may be purified to substantial purity by standard techniques, including selective precipitation with such substances as ammonium sulfate; column chromatography, immunopurification methods, and others (see, e.g., Scopes, Protein Purification: Principles and Practice (1982 ); U.S. Patent No. 4,673,641 ; Ausubel et al., supra; and Sambrook et al., supra ).
  • polypeptides having established molecular adhesion properties can be reversible fused to polypeptides of the invention.
  • the polypeptides can be selectively adsorbed to a purification column and then freed from the column in a relatively pure form. The fused protein is then removed by enzymatic activity. Finally the polypeptide can be purified using immunoaffinity columns.
  • inclusion bodies When recombinant proteins are expressed by the transformed bacteria in large amounts, typically after promoter induction, although expression can be constitutive, the proteins may form insoluble aggregates.
  • purification of protein inclusion bodies typically involves the extraction, separation and/or purification of inclusion bodies by disruption of bacterial cells typically, but not limited to, by incubation in a buffer of about 100-150 ⁇ g/ml lysozyme and 0.1% Nonidet P40, a non-ionic detergent.
  • the cell suspension can be ground using a Polytron grinder (Brinkman Instruments, Westbury, NY). Alternatively, the cells can be sonicated on ice. Alternate methods of lysing bacteria are described in Ausubel et al. and Sambrook et al., both supra, and will be apparent to those of skill in the art.
  • the cell suspension is generally centrifuged and the pellet containing the inclusion bodies resuspended in buffer which does not dissolve but washes the inclusion bodies, e.g., 20 mM Tris-HCl (pH 7.2), 1 mM EDTA, 150 mM NaCl and 2% Triton-X 100, a non-ionic detergent. It may be necessary to repeat the wash step to remove as much cellular debris as possible.
  • the remaining pellet of inclusion bodies may be resuspended in an appropriate buffer (e.g., 20 mM sodium phosphate, pH 6.8, 150 mM NaCl).
  • an appropriate buffer e.g., 20 mM sodium phosphate, pH 6.8, 150 mM NaCl.
  • Other appropriate buffers will be apparent to those of skill in the art.
  • the inclusion bodies are solubilized by the addition of a solvent that is both a strong hydrogen acceptor and a strong hydrogen donor (or a combination of solvents each having one of these properties).
  • a solvent that is both a strong hydrogen acceptor and a strong hydrogen donor or a combination of solvents each having one of these properties.
  • the proteins that formed the inclusion bodies may then be renatured by dilution or dialysis with a compatible buffer.
  • Suitable solvents include, but are not limited to, urea (from about 4 M to about 8 M), formamide (at least about 80%, volume/volume basis), and guanidine hydrochloride (from about 4 M to about 8 M).
  • Some solvents that are capable of solubilizing aggregate-forming proteins are inappropriate for use in this procedure due to the possibility of irreversible denaturation of the proteins, accompanied by a lack of immunogenicity and/or activity.
  • SDS sodium dodecyl sulfate
  • 70% formic acid Some solvents that are capable of solubilizing aggregate-forming proteins, such as SDS (sodium dodecyl sulfate) and 70% formic acid, are inappropriate for use in this procedure due to the possibility of irreversible denaturation of the proteins, accompanied by a lack of immunogenicity and/or activity.
  • guanidine hydrochloride and similar agents are denaturants, this denaturation is not irreversible and renaturation may occur upon removal (by dialysis, for example) or dilution of the denaturant, allowing re-formation of the immunologically and/or biologically active protein of interest.
  • the protein can be separated from other bacterial proteins by standard separation techniques.
  • the periplasmic fraction of the bacteria can be isolated by cold osmotic shock in addition to other methods known to those of skill in the art (see, Ausubel et al., supra).
  • the bacterial cells are centrifuged to form a pellet. The pellet is resuspended in a buffer containing 20% sucrose.
  • the bacteria are centrifuged and the pellet is resuspended in ice-cold 5 mM MgSO 4 and kept in an ice bath for approximately 10 minutes.
  • the cell suspension is centrifuged and the supernatant decanted and saved.
  • the recombinant proteins present in the supernatant can be separated from the host proteins by standard separation techniques well known to those of skill in the art.
  • an initial salt fractionation can separate many of the unwanted host cell proteins (or proteins derived from the cell culture media) from the recombinant protein of interest.
  • the preferred salt is ammonium sulfate.
  • Ammonium sulfate precipitates proteins by effectively reducing the amount of water in the protein mixture. Proteins then precipitate on the basis of their solubility. The more hydrophobic a protein is, the more likely it is to precipitate at lower ammonium sulfate concentrations.
  • a typical protocol is to add saturated ammonium sulfate to a protein solution so that the resultant ammonium sulfate concentration is between 20-30%. This will precipitate the most hydrophobic proteins.
  • the precipitate is discarded (unless the protein of interest is hydrophobic) and ammonium sulfate is added to the supernatant to a concentration known to precipitate the protein of interest.
  • the precipitate is then solubilized in buffer and the excess salt removed if necessary, through either dialysis or diafiltration.
  • Other methods that rely on solubility of proteins, such as cold ethanol precipitation, are well known to those of skill in the art and can be used to fractionate complex protein mixtures.
  • a protein of greater and lesser size can be isolated using ultrafiltration through membranes of different pore sizes (for example, Amicon or Millipore membranes).
  • the protein mixture is ultrafiltered through a membrane with a pore size that has a lower molecular weight cut-off than the molecular weight of the protein of interest.
  • the retentate of the ultrafiltration is then ultrafiltered against a membrane with a molecular cut off greater than the molecular weight of the protein of interest.
  • the recombinant protein will pass through the membrane into the filtrate.
  • the filtrate can then be chromatographed as described below.
  • proteins of interest can also be separated from other proteins on the basis of their size, net surface charge, hydrophobicity and affinity for ligands.
  • antibodies raised against proteins can be conjugated to column matrices and the proteins immunopurified. All of these methods are well known in the art.
  • detection of expression of polynucleotides of the invention has many uses. For example, as discussed herein, detection of the level of polypeptides or polynucleotides of the invention in a patient is useful for diagnosing mood disorders or psychotic disorders or a predisposition for a mood disorder or psychotic disorders. Moreover, detection of gene expression is useful to identify modulators of expression of the polypeptides or polynucleotides of the invention.
  • DNA and RNA measurement using nucleic acid hybridization techniques are known to those of skill in the art (see, Sambrook, supra). Some methods involve an electrophoretic separation (e.g., Southern blot for detecting DNA, and Northern blot for detecting RNA), but measurement of DNA and RNA can also be carried out in the absence of electrophoretic separation (e.g., by dot blot). Southern blot of genomic DNA (e.g., from a human) can be used for screening for restriction fragment length polymorphism (RFLP) to detect the presence of a genetic disorder affecting a polypeptide of the invention.
  • RFLP restriction fragment length polymorphism
  • nucleic acid hybridization format is not critical.
  • a variety of nucleic acid hybridization formats are known to those skilled in the art.
  • common formats include sandwich assays and competition or displacement assays.
  • Hybridization techniques are generally described in Hames and Higgins Nucleic Acid Hybridization, A Practical Approach, IRL Press (1985 ); Gall and Pardue, Proc. Natl. Acad. Sci. U.S.A., 63:378-383 (1969 ); and John et al. Nature, 223:582-587 (1969 ).
  • Detection of a hybridization complex may require the binding of a signal-generating complex to a duplex of target and probe polynucleotides or nucleic acids. Typically, such binding occurs through ligand and anti-ligand interactions as between a ligand-conjugated probe and an anti-ligand conjugated with a signal.
  • the binding of the signal generation complex is also readily amenable to accelerations by exposure to ultrasonic energy.
  • the label may also allow indirect detection of the hybridization complex.
  • the label is a hapten or antigen
  • the sample can be detected by using antibodies.
  • a signal is generated by attaching fluorescent or enzyme molecules to the antibodies or in some cases, by attachment to a radioactive label (see, e.g., Tijssen, "Practice and Theory of Enzyme Immunoassays," Laboratory Techniques in Biochemistry and Molecular Biology, Burdon and van Knippenberg Eds., Elsevier (1985), pp. 9-20 ).
  • the probes are typically labeled either directly, as with isotopes, chromophores, lumiphores, chromogens, or indirectly, such as with biotin, to which a streptavidin complex may later bind.
  • the detectable labels used in the assays of the present invention can be primary labels (where the label comprises an element that is detected directly or that produces a directly detectable element) or secondary labels (where the detected label binds to a primary label, e.g., as is common in immunological labeling).
  • labeled signal nucleic acids are used to detect hybridization.
  • Complementary nucleic acids or signal nucleic acids may be labeled by any one of several methods typically used to detect the presence of hybridized polynucleotides. The most common method of detection is the use of autoradiography with 3 H, 125 I, 35 S, 14 C, or 32 P-labeled probes or the like.
  • labels include, e.g., ligands that bind to labeled antibodies, fluorophores, chemiluminescent agents, enzymes, and antibodies which can serve as specific binding pair members for a labeled ligand.
  • ligands that bind to labeled antibodies, fluorophores, chemiluminescent agents, enzymes, and antibodies which can serve as specific binding pair members for a labeled ligand.
  • An introduction to labels, labeling procedures and detection of labels is found in Polak and Van Noorden Introduction to Immunocytochemistry, 2nd ed., Springer Verlag, NY (1997 ); and in Haugland Handbook of Fluorescent Probes and Research Chemicals, a combined handbook and catalogue Published by Molecular Probes, Inc. (1996 ).
  • a detector which monitors a particular probe or probe combination is used to detect the detection reagent label.
  • Typical detectors include spectrophotometers, phototubes and photodiodes, microscopes, scintillation counters, cameras, film and the like, as well as combinations thereof. Examples of suitable detectors are widely available from a variety of commercial sources known to persons of skill in the art. Commonly, an optical image of a substrate comprising bound labeling moieties is digitized for subsequent computer analysis.
  • the amount of RNA is measured by quantifying the amount of label fixed to the solid support by binding of the detection reagent.
  • the presence of a modulator during incubation will increase or decrease the amount of label fixed to the solid support relative to a control incubation which does not comprise the modulator, or as compared to a baseline established for a particular reaction type.
  • Means of detecting and quantifying labels are well known to those of skill in the art.
  • the target nucleic acid or the probe is immobilized on a solid support.
  • Solid supports suitable for use in the assays of the invention are known to those of skill in the art. As used herein, a solid support is a matrix of material in a substantially fixed arrangement.
  • VLSIPSTM very large scale immobilized polymer arrays
  • Affymetrix, Inc. (Santa Clara, CA) can be used to detect changes in expression levels of a plurality of genes involved in the same regulatory pathways simultaneously. See, Tijssen, supra., Fodor et al. (1991) Science, 251: 767- 777 ; Sheldon et al. (1993) Clinical Chemistry 39(4): 718-719 , and Kozal et al. (1996) Nature Medicine 2(7): 753-759 .
  • Detection can be accomplished, for example, by using a labeled detection moiety that binds specifically to duplex nucleic acids (e.g., an antibody that is specific for RNA-DNA duplexes).
  • a labeled detection moiety that binds specifically to duplex nucleic acids
  • a labeled detection moiety that binds specifically to duplex nucleic acids
  • One preferred example uses an antibody that recognizes DNA-RNA heteroduplexes in which the antibody is linked to an enzyme (typically by recombinant or covalent chemical bonding). The antibody is detected when the enzyme reacts with its substrate, producing a detectable product.
  • the nucleic acids used in this invention can be either positive or negative probes. Positive probes bind to their targets and the presence of duplex formation is evidence of the presence of the target. Negative probes fail to bind to the suspect target and the absence of duplex formation is evidence of the presence of the target.
  • the use of a wild type specific nucleic acid probe or PCR primers may serve as a negative probe in an assay sample where only the nucleotide sequence of interest is present.
  • the sensitivity of the hybridization assays may be enhanced through use of a nucleic acid amplification system that multiplies the target nucleic acid being detected.
  • a nucleic acid amplification system that multiplies the target nucleic acid being detected.
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • Other methods recently described in the art are the nucleic acid sequence based amplification (NASBA, Cangene, Mississauga, Ontario) and Q Beta Replicase systems. These systems can be used to directly identify mutants where the PCR or LCR primers are designed to be extended or ligated only when a selected sequence is present.
  • the selected sequences can be generally amplified using, for example, nonspecific PCR primers and the amplified target region later probed for a specific sequence indicative of a mutation.
  • An alternative means for determining the level of expression of the nucleic acids of the present invention is in situ hybridization.
  • In situ hybridization assays are well known and are generally described in Angerer et al., Methods Enzymol. 152:649-660 (1987 ).
  • cells preferentially human cells from the cerebellum or the hippocampus, are fixed to a solid support, typically a glass slide. If DNA is to be probed, the cells are denatured with heat or alkali. The cells are then contacted with a hybridization solution at a moderate temperature to permit annealing of specific probes that are labeled.
  • the probes are preferably labeled with radioisotopes or fluorescent reporters.
  • Immunoassays can be used to qualitatively or quantitatively analyze polypeptides. A general overview of the applicable technology can be found in Harlow & Lane, Antibodies: A Laboratory Manual (1988 ).
  • a recombinant protein is produced in a transformed cell line.
  • An inbred strain of mice or rabbits is immunized with the protein using a standard adjuvant, such as Freund's adjuvant, and a standard immunization protocol.
  • a synthetic peptide derived from the sequences disclosed herein and conjugated to a carrier protein can be used as an immunogen.
  • Polyclonal sera are collected and titered against the immunogen in an immunoassay, for example, a solid phase immunoassay with the immunogen immobilized on a solid support.
  • Polyclonal antisera with a titer of 10 4 or greater are selected and tested for their cross-reactivity against unrelated proteins or even other homologous proteins from other organisms, using a competitive binding immunoassay.
  • Specific monoclonal and polyclonal antibodies and antisera will usually bind with a K D of at least about 0.1 mM, more usually at least about 1 ⁇ M, preferably at least about 0.1 ⁇ M or better, and most preferably, 0.01 ⁇ M or better.
  • a number of proteins of the invention comprising immunogens may be used to produce antibodies specifically or selectively reactive with the proteins of interest.
  • Recombinant protein is the preferred immunogen for the production of monoclonal or polyclonal antibodies.
  • Naturally occurring protein such as one comprising an amino acid sequence encoded by a gene listed in Table 1-8 may also be used either in pure or impure form.
  • Synthetic peptides made using the protein sequences described herein may also be used as an immunogen for the production of antibodies to the protein.
  • Recombinant protein can be expressed in eukaryotic or prokaryotic cells and purified as generally described supra. The product is then injected into an animal capable of producing antibodies. Either monoclonal or polyclonal antibodies may be generated for subsequent use in immunoassays to measure the protein.
  • an immunogen preferably a purified protein
  • an adjuvant preferably an adjuvant
  • animals are immunized.
  • the animal's immune response to the immunogen preparation is monitored by taking test bleeds and determining the titer of reactivity to the polypeptide of interest.
  • blood is collected from the animal and antisera are prepared. Further fractionation of the antisera to enrich for antibodies reactive to the protein can be done if desired (see, Harlow and Lane, supra).
  • Monoclonal antibodies may be obtained using various techniques familiar to those of skill in the art.
  • spleen cells from an animal immunized with a desired antigen are immortalized, commonly by fusion with a myeloma cell (see, Kohler and Milstein, Eur. J. Immunol. 6:511-519 (1976 )).
  • Alternative methods of immortalization include, e.g., transformation with Epstein Barr Virus, oncogenes, or retroviruses, or other methods well known in the art.
  • Colonies arising from single immortalized cells are screened for production of antibodies of the desired specificity and affinity for the antigen, and yield of the monoclonal antibodies produced by such cells may be enhanced by various techniques, including injection into the peritoneal cavity of a vertebrate host.
  • the protein can be measured by a variety of immunoassay methods with qualitative and quantitative results available to the clinician.
  • immunoassays of the present invention can be performed in any of several configurations, which are reviewed extensively in Maggio Enzyme Immunoassay, CRC Press, Boca Raton, Florida (1980 ); Tijssen, supra; and Harlow and Lane, supra.
  • Immunoassays to measure target proteins in a human sample may use a polyclonal antiserum that was raised to the protein (e.g., one has an amino acid sequence encoded by a gene listed in Table 1-8) or a fragment thereof. This antiserum is selected to have low cross-reactivity against different proteins and any such cross-reactivity is removed by immunoabsorption prior to use in the immunoassay.
  • a protein of interest is detected and/or quantified using any of a number of well-known immunological binding assays (see, e.g., U.S. Patents 4,366,241 ; 4,376,110 ; 4,517,288 ; and 4,837,168 ).
  • immunological binding assays see also Asai Methods in Cell Biology Volume 37: Antibodies in Cell Biology, Academic Press, Inc. NY (1993 ); Stites, supra.
  • Immunological binding assays or immunoassays typically utilize a "capture agent" to specifically bind to and often immobilize the analyte (in this case a polypeptide of the present invention or antigenic subsequences thereof).
  • the capture agent is a moiety that specifically binds to the analyte.
  • the capture agent is an antibody that specifically binds, for example, a polypeptide of the invention.
  • the antibody may be produced by any of a number of means well known to those of skill in the art and as described above.
  • Immunoassays also often utilize a labeling agent to specifically bind to and label the binding complex formed by the capture agent and the analyte.
  • the labeling agent may itself be one of the moieties comprising the antibody/analyte complex.
  • the labeling agent may be a third moiety, such as another antibody, that specifically binds to the antibody/protein complex.
  • the labeling agent is a second antibody bearing a label.
  • the second antibody may lack a label, but it may, in turn, be bound by a labeled third antibody specific to antibodies of the species from which the second antibody is derived.
  • the second antibody can be modified with a detectable moiety, such as biotin, to which a third labeled molecule can specifically bind, such as enzyme-labeled streptavidin.
  • proteins capable of specifically binding immunoglobulin constant regions can also be used as the label agents. These proteins are normal constituents of the cell walls of streptococcal bacteria. They exhibit a strong non-immunogenic reactivity with immunoglobulin constant regions from a variety of species (see, generally, Kronval, et al. J. Immunol., 111:1401-1406 (1973 ); and Akerstrom, et al. J. Immunol., 135:2589-2542 (1985 )).
  • incubation and/or washing steps may be required after each combination of reagents. Incubation steps can vary from about 5 seconds to several hours, preferably from about 5 minutes to about 24 hours. The incubation time will depend upon the assay format, analyte, volume of solution, concentrations, and the like. Usually, the assays will be carried out at ambient temperature, although they can be conducted over a range of temperatures, such as 10°C to 40°C.
  • Immunoassays for detecting proteins of interest from tissue samples may be either competitive or noncompetitive.
  • Noncompetitive immunoassays are assays in which the amount of captured analyte (in this case the protein) is directly measured.
  • the capture agent e.g., antibodies specific for a polypeptide encoded by a gene listed in Table 1-8
  • the capture agent can be bound directly to a solid substrate where it is immobilized. These immobilized antibodies then capture the polypeptide present in the test sample.
  • the polypeptide thus immobilized is then bound by a labeling agent, such as a second antibody bearing a label.
  • the second antibody may lack a label, but it may, in turn, be bound by a labeled third antibody specific to antibodies of the species from which the second antibody is derived.
  • the second can be modified with a detectable moiety, such as biotin, to which a third labeled molecule can specifically bind, such as enzyme-labeled streptavidin.
  • the amount of analyte (such as a polypeptide encoded by a gene listed in Table 1-8) present in the sample is measured indirectly by measuring the amount of an added (exogenous) analyte displaced (or competed away) from a capture agent (e.g., an antibody specific for the analyte) by the analyte present in the sample.
  • a capture agent e.g., an antibody specific for the analyte
  • the antibody is immobilized on a solid substrate.
  • the amount of the polypeptide bound to the antibody may be determined either by measuring the amount of subject protein present in a protein/antibody complex or, alternatively, by measuring the amount of remaining uncomplexed protein.
  • the amount of protein may be detected by providing a labeled protein molecule.
  • Immunoassays in the competitive binding format can be used for cross-reactivity determinations.
  • a protein of interest can be immobilized on a solid support. Proteins are added to the assay which compete with the binding of the antisera to the immobilized antigen. The ability of the above proteins to compete with the binding of the antisera to the immobilized protein is compared to that of the protein of interest. The percent cross-reactivity for the above proteins is calculated, using standard calculations. Those antisera with less than 10% cross-reactivity with each of the proteins listed above are selected and pooled. The cross-reacting antibodies are optionally removed from the pooled antisera by immunoabsorption with the considered proteins, e.g., distantly related homologs.
  • the immunoabsorbed and pooled antisera are then used in a competitive binding immunoassay as described above to compare a second protein, thought to be perhaps a protein of the present invention, to the immunogen protein.
  • the two proteins are each assayed at a wide range of concentrations and the amount of each protein required to inhibit 50% of the binding of the antisera to the immobilized protein is determined. If the amount of the second protein required is less than 10 times the amount of the protein partially encoded by a sequence herein that is required, then the second protein is said to specifically bind to an antibody generated to an immunogen consisting of the target protein.
  • western blot (immunoblot) analysis is used to detect and quantify the presence of a polypeptide of the invention in the sample.
  • the technique generally comprises separating sample proteins by gel electrophoresis on the basis of molecular weight, transferring the separated proteins to a suitable solid support (such as, e.g., a nitrocellulose filter, a nylon filter, or a derivatized nylon filter) and incubating the sample with the antibodies that specifically bind the protein of interest.
  • the antibodies specifically bind to a polypeptide of interest on the solid support.
  • These antibodies may be directly labeled or alternatively may be subsequently detected using labeled antibodies (e.g., labeled sheep anti-mouse antibodies) that specifically bind to the antibodies against the protein of interest.
  • LISA liposome immunoassays
  • the particular label or detectable group used in the assay is not a critical aspect of the invention, as long as it does not significantly interfere with the specific binding of the antibody used in the assay.
  • the detectable group can be any material having a detectable physical or chemical property.
  • Such detectable labels have been well developed in the field of immunoassays and, in general, most labels useful in such methods can be applied to the present invention.
  • a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.
  • Useful labels in the present invention include magnetic beads (e.g., Dynabeads TM ), fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the like), radiolabels (e.g., 3 H, 125 I, 35 S, 14 C, or 32 P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads.
  • fluorescent dyes e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the like
  • radiolabels e.g., 3 H, 125 I, 35 S, 14 C, or 32 P
  • enzymes e.g., horse radish peroxidase, alkaline phosphatase
  • the label may be coupled directly or indirectly to the desired component of the assay according to methods well known in the art. As indicated above, a wide variety of labels may be used, with the choice of label depending on the sensitivity required, the ease of conjugation with the compound, stability requirements, available instrumentation, and disposal provisions.
  • Non-radioactive labels are often attached by indirect means.
  • the molecules can also be conjugated directly to signal generating compounds, e.g., by conjugation with an enzyme or fluorescent compound.
  • an enzyme or fluorescent compound e.g., A variety of enzymes and fluorescent compounds can be used with the methods of the present invention and are well-known to those of skill in the art (for a review of various labeling or signal producing systems which may be used, see, e.g., U.S. Patent No. 4,391,904 ).
  • Means of detecting labels are well known to those of skill in the art.
  • means for detection include a scintillation counter or photographic film as in autoradiography.
  • the label is a fluorescent label, it may be detected by exciting the fluorochrome with the appropriate wavelength of light and detecting the resulting fluorescence. The fluorescence may be detected visually, by means of photographic film, by the use of electronic detectors such as charge-coupled devices (CCDs) or photomultipliers and the like.
  • CCDs charge-coupled devices
  • enzymatic labels may be detected by providing the appropriate substrates for the enzyme and detecting the resulting reaction product.
  • simple colorimetric labels may be detected directly by observing the color associated with the label. Thus, in various dipstick assays, conjugated gold often appears pink, while various conjugated beads appear the color of the bead.
  • agglutination assays can be used to detect the presence of the target antibodies.
  • antigen-coated particles are agglutinated by samples comprising the target antibodies.
  • none of the components need to be labeled and the presence of the target antibody is detected by simple visual inspection.
  • BP or MDD in a patient may be diagnosed or otherwise evaluated by visualizing expression in situ of one or more of the appropriately dysregulated gene sequences identified herein.
  • visualizing expression in situ of one or more of the appropriately dysregulated gene sequences identified herein Those skilled in the art of visualizing the presence or expression of molecules including nucleic acids, polypeptides and other biochemicals in the brains of living patients will appreciate that the gene expression information described herein may be utilized in the context of a variety of visualization methods. Such methods include, but are not limited to, single-photon emission-computed tomography (SPECT) and positron-emitting tomography (PET) methods. See, e.g., Vassaux and Groot-wassink, "In Vivo Noninvasive Imaging for Gene Therapy," J. Biomedicine and Biotechnology, 2: 92-101 (2003 ).
  • SPECT single-photon emission-computed tomography
  • PET positron-emitting tomography
  • PET and SPECT imaging shows the chemical functioning of organs and tissues, while other imaging techniques - such as X-ray, CT and MRI - show structure.
  • the use of PET and SPECT imaging is useful for qualifying and monitoring the development of brain diseases, including schizophrenia and related disorders.
  • the use of PET or SPECT imaging allows diseases to be detected years earlier than the onset of symptoms.
  • the dysregulated genes disclosed in Tables 1-30 can be used in the context of PET and SPECT imaging applications. After modification with appropriate tracer residues for PET or SPECT applications, molecules which interact or bind with the transcripts in Tables 1-30 or with any polypeptides encoded by those transcripts may be used to visualize the patterns of gene expression and facilitate diagnosis of schizophrenia MDD or BP, as described herein. Similarly, if the encoded polypeptides encode enzymes, labeled molecules which interact with the products of catalysis by the enzyme may be used for the in vivo imaging and diagnostic application described herein.
  • Antisense technology is particularly suitable for detecting the the transcripts identified in Tables 1-30 herein.
  • PNA antisense peptide nucleic acid
  • an appropriate radionuclide such as 111 In
  • Suzuki et al. utilize a delivery system comprising monoclonal antibodies that target transferring receptors at the blood-brain barrier and facilitate transport of the PNA across that barrier.
  • Modified embodiments of this technique may be used to target upregulated genes associated with schizophrenia, BP or MDD, such as the upregulated genes which appear in Tables 1-30, in methods of treating schizophrenic, BP or MDD patients.
  • the dysregulated genes listed in Tables 1-30 may be used in the context of prenatal and neonatal diagnostic methods. For example, fetal or neonatal samples can be obtained and the expression levels of appropriate transcripts (e.g ., the transcripts in Table 19) may be measured and correlated with the presence or increased likelihood of a mental disorder, e.g ., MDD. Similarly, the presence of one or more of the SNPs identified in the Tables, e.g ., Table 27 may be used to infer or corroborate dysregulated expression of a gene and the likelihood of a mood disorder in prenatal, neonatal, children and adult patients.
  • appropriate transcripts e.g ., the transcripts in Table 19
  • the presence of one or more of the SNPs identified in the Tables e.g ., Table 27 may be used to infer or corroborate dysregulated expression of a gene and the likelihood of a mood disorder in prenatal, neonatal, children and adult patients.
  • the brain labeling and imaging techniques described herein or variants thereof may be used in conjunction with any of the dysregulated gene sequences in Tables 1-30 in a forensic analysis, i.e ., to determine whether a deceased individual suffered from schizophrenia, BP, or MDD.
  • Modulators of polypeptides or polynucleotides of the invention i.e. agonists or antagonists of their activity or modulators of polypeptide or polynucleotide expression, are useful for treating a number of human diseases, including mood disorders or psychotic disorders.
  • Administration of agonists, antagonists or other agents that modulate expression of the polynucleotides or polypeptides of the invention can be used to treat patients with mood disorders or psychotic disorders.
  • screening protocols can be utilized to identify agents that modulate the level of expression or activity of polypeptides and polynucleotides of the invention in cells, particularly mammalian cells, and especially human cells.
  • the screening methods involve screening a plurality of agents to identify an agent that modulates the polypeptide activity by binding to a polypeptide of the invention, modulating inhibitor binding to the polypeptide or activating expression of the polypeptide or polynucleotide, for example.
  • Preliminary screens can be conducted by screening for agents capable of binding to a polypeptide of the invention, as at least some of the agents so identified are likely modulators of polypeptide activity.
  • the binding assays usually involve contacting a polypeptide of the invention with one or more test agents and allowing sufficient time for the protein and test agents to form a binding complex. Any binding complexes formed can be detected using any of a number of established analytical techniques.
  • Protein binding assays include, but are not limited to, methods that measure co-precipitation, co-migration on non-denaturing SDS-polyacrylamide gels, and co-migration on Western blots ( see, e.g., Bennet and Yamamura, (1985) "Neurotransmitter, Hormone or Drug Receptor Binding Methods," in Neurotransmitter Receptor Binding (Yamamura, H. I., et al., eds.), pp. 61-89 .
  • the protein utilized in such assays can be naturally expressed, cloned or synthesized.
  • Binding assays are also useful, e.g., for identifying endogenous proteins that interact with a polypeptide of the invention.
  • binding assays e.g., antibodies, receptors or other molecules that bind a polypeptide of the invention can be identified in binding assays.
  • Certain screening methods involve screening for a compound that up or down-regulates the expression of a polypeptide or polynucleotide of the invention.
  • Such methods generally involve conducting cell-based assays in which test compounds are contacted with one or more cells expressing a polypeptide or polynucleotide of the invention and then detecting an increase or decrease in expression (either transcript, translation product, or catalytic product).
  • Some assays are performed with peripheral cells, or other cells, that express an endogenous polypeptide or polynucleotide of the invention.
  • Polypeptide or polynucleotide expression can be detected in a number of different ways.
  • the expression level of a polynucleotide of the invention in a cell can be determined by probing the mRNA expressed in a cell with a probe that specifically hybridizes with a transcript (or complementary nucleic acid derived therefrom) of a polynucleotide of the invention. Probing can be conducted by lysing the cells and conducting Northern blots or without lysing the cells using in situ-hybridization techniques.
  • a polypeptide of the invention can be detected using immunological methods in which a cell lysate is probed with antibodies that specifically bind to a polypeptide of the invention.
  • reporter assays conducted with cells that do not express a polypeptide or polynucleotide of the invention. Certain of these assays are conducted with a heterologous nucleic acid construct that includes a promoter of a polynucleotide of the invention that is operably linked to a reporter gene that encodes a detectable product.
  • reporter genes can be utilized. Some reporters are inherently detectable. An example of such a reporter is green fluorescent protein that emits fluorescence that can be detected with a fluorescence detector. Other reporters generate a detectable product. Often such reporters are enzymes.
  • Exemplary enzyme reporters include, but are not limited to, ⁇ -glucuronidase, chloramphenicol acetyl transferase (CAT); Alton and Vapnek (1979) Nature 282:864-869 ), luciferase, ⁇ -galactosidase, green fluorescent protein (GFP) and alkaline phosphatase ( Toh, et al. (1980) Eur. J. Biochem. 182:231-238 ; and Hall et al. (1983) J. Mol. Appl. Gen. 2:101 ).
  • CAT chloramphenicol acetyl transferase
  • GFP green fluorescent protein
  • alkaline phosphatase Toh, et al. (1980) Eur. J. Biochem. 182:231-238 ; and Hall et al. (1983) J. Mol. Appl. Gen. 2:101 ).
  • cells harboring the reporter construct are contacted with a test compound.
  • a test compound that either activates the promoter by binding to it or triggers a cascade that produces a molecule that activates the promoter causes expression of the detectable reporter.
  • Certain other reporter assays are conducted with cells that harbor a heterologous construct that includes a transcriptional control element that activates expression of a polynucleotide of the invention and a reporter operably linked thereto.
  • an agent that binds to the transcriptional control element to activate expression of the reporter or that triggers the formation of an agent that binds to the transcriptional control element to activate reporter expression can be identified by the generation of signal associated with reporter expression.
  • the level of expression or activity can be compared to a baseline value.
  • the baseline value can be a value for a control sample or a statistical value that is representative of expression levels for a control population (e.g., healthy individuals not having or at risk for mood disorders or psychotic disorders).
  • Expression levels can also be determined for cells that do not express a polynucleotide of the invention as a negative control. Such cells generally are otherwise substantially genetically the same as the test cells.
  • Cells that express an endogenous polypeptide or polynucleotide of the invention include, e.g., brain cells, including cells from the cerebellum, anterior cingulate cortex, dorsolateral prefrontal cortex, amygdala, hippocampus, or nucleus accumbens.
  • Cells that do not endogenously express polynucleotides of the invention can be prokaryotic, but are preferably eukaryotic.
  • the eukaryotic cells can be any of the cells typically utilized in generating cells that harbor recombinant nucleic acid constructs.
  • Exemplary eukaryotic cells include, but are not limited to, yeast, and various higher eukaryotic cells such as the COS, CHO and HeLa cell lines.
  • Catalytic activity of polypeptides of the invention can be determined by measuring the production of enzymatic products or by measuring the consumption of substrates. Activity refers to either the rate of catalysis or the ability to the polypeptide to bind (K m ) the substrate or release the catalytic product (K d ).
  • polypeptides of the invention are performed according to general biochemical analyses.
  • assays include cell-based assays as well as in vitro assays involving purified or partially purified polypeptides or crude cell lysates.
  • the assays generally involve providing a known quantity of substrate and quantifying product as a function of time.
  • Agents that are initially identified by any of the foregoing screening methods can be further tested to validate the apparent activity.
  • Such studies are conducted with suitable animal models.
  • the basic format of such methods involves administering a lead compound identified during an initial screen to an animal that serves as a model for humans and then determining if expression or activity of a polynucleotide or polypeptide of the invention is in fact upregulated.
  • the animal models utilized in validation studies generally are mammals of any kind. Specific examples of suitable animals include, but are not limited to, primates, mice, and rats. As described herein, models using admininstration of known therapeutics can be useful.
  • Animal models of mental disorders also find use in screening for modulators.
  • invertebrate models such as Drosophila models can be used, screening for modulators of Drosophila orthologs of the human genes disclosed herein.
  • transgenic animal technology including gene knockout technology, for example as a result of homologous recombination with an appropriate gene targeting vector, or gene overexpression, will result in the absence, decreased or increased expression of a polynucleotide or polypeptide of the invention.
  • the same technology can also be applied to make knockout cells.
  • tissue-specific expression or knockout of a polynucleotide or polypeptide of the invention may be necessary.
  • Transgenic animals generated by such methods find use as animal models of mental illness and are useful in screening for modulators of mental illness.
  • Knockout cells and transgenic mice can be made by insertion of a marker gene or other heterologous gene into an endogenous gene site in the mouse genome via homologous recombination. Such mice can also be made by substituting an endogenous polynucleotide of the invention with a mutated version of the polynucleotide, or by mutating an endogenous polynucleotide, e.g., by exposure to carcinogens.
  • a DNA construct is introduced into the nuclei of embryonic stem cells.
  • Cells containing the newly engineered genetic lesion are injected into a host mouse embryo, which is re-implanted into a recipient female. Some of these embryos develop into chimeric mice that possess germ cells partially derived from the mutant cell line. Therefore, by breeding the chimeric mice it is possible to obtain a new line of mice containing the introduced genetic lesion ( see, e.g., Capecchi et al., Science 244:1288 (1989 )).
  • Chimeric targeted mice can be derived according to Hogan et al., Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory (1988 ) and Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, Robertson, ed., IRL Press, Washington, D.C., (1987 ).
  • the agents tested as modulators of the polypeptides or polynucleotides of the invention can be any small chemical compound, or a biological entity, such as a protein, sugar, nucleic acid or lipid.
  • modulators can be genetically altered versions of a polypeptide or polynucleotide of the invention.
  • test compounds will be small chemical molecules and peptides.
  • any chemical compound can be used as a potential modulator or ligand in the assays of the invention, although most often compounds that can be dissolved in aqueous or organic (especially DMSO-based) solutions are used.
  • the assays are designed to screen large chemical libraries by automating the assay steps and providing compounds from any convenient source to assays, which are typically run in parallel (e.g., in microtiter formats on microtiter plates in robotic assays). It will be appreciated that there are many suppliers of chemical compounds, including Sigma (St. Louis, MO), Aldrich (St. Louis, MO), Sigma-Aldrich (St. Louis, MO), Fluka Chemika-Biochemica Analytika (Buchs, Switzerland) and the like. Modulators also include agents designed to reduce the level of mRNA of the invention (e.g. antisense molecules, ribozymes, DNAzymes and the like) or the level of translation from an mRNA.
  • mRNA of the invention e.g. antisense molecules, ribozymes, DNAzymes and the like
  • high throughput screening methods involve providing a combinatorial chemical or peptide library containing a large number of potential therapeutic compounds (potential modulator or ligand compounds). Such "combinatorial chemical libraries” or “ligand libraries” are then screened in one or more assays, as described herein, to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity. The compounds thus identified can serve as conventional "lead compounds” or can themselves be used as potential or actual therapeutics.
  • a combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis, by combining a number of chemical "building blocks” such as reagents.
  • a linear combinatorial chemical library such as a polypeptide library is formed by combining a set of chemical building blocks (amino acids) in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks.
  • combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Patent 5,010,175 , Furka, Int. J. Pept. Prot. Res. 37:487-493 (1991 ) and Houghton et al., Nature 354:84-88 (1991 )).
  • chemistries for generating chemical diversity libraries can also be used. Such chemistries include, but are not limited to: peptoids (e.g., PCT Publication No.
  • WO 91/19735 encoded peptides (e.g., PCT Publication WO 93/20242 ), random bio-oligomers (e.g., PCT Publication No. WO 92/00091 ), benzodiazepines (e.g., U.S. Pat. No. 5,288,514 ), diversomers such as hydantoins, benzodiazepines and dipeptides ( Hobbs et al., Proc. Nat. Acad. Sci. USA 90:6909-6913 (1993 )), vinylogous polypeptides ( Hagihara et al., J. Amer. Chem. Soc.
  • nucleic acid libraries see Ausubel, Berger and Sambrook, all supra
  • peptide nucleic acid libraries see, e.g., U.S. Patent 5,539,083
  • antibody libraries see, e.g., Vaughn et al., Nature Biotechnology, 14(3):309-314 (1996 ) and PCT/US96/10287
  • carbohydrate libraries see, e.g., Liang et al., Science, 274:1520-1522 (1996 ) and U.S.
  • Patent 5,593,853 small organic molecule libraries (see, e.g., benzodiazepines, Baum C&EN, Jan 18, page 33 (1993 ); isoprenoids, U.S. Patent 5,569,588 ; thiazolidinones and metathiazanones, U.S. Patent 5,549,974 ; pyrrolidines, U.S. Patents 5,525,735 and 5,519,134 ; morpholino compounds, U.S. Patent 5,506,337 ; benzodiazepines, 5,288,514 , and the like).
  • small organic molecule libraries see, e.g., benzodiazepines, Baum C&EN, Jan 18, page 33 (1993 ); isoprenoids, U.S. Patent 5,569,588 ; thiazolidinones and metathiazanones, U.S. Patent 5,549,974 ; pyrrolidines, U.S. Patents 5,525,735 and 5,519,134
  • each well of a microtiter plate can be used to run a separate assay against a selected potential modulator, or, if concentration or incubation time effects are to be observed, every 5-10 wells can test a single modulator.
  • a single standard microtiter plate can assay about 100 (e.g., 96) modulators. If 1536 well plates are used, then a single plate can easily assay from about 100 to about 1500 different compounds. It is possible to assay several different plates per day; assay screens for up to about 6,000-20,000 different compounds are possible using the integrated systems of the invention. More recently, microfluidic approaches to reagent manipulation have been developed.
  • the molecule of interest can be bound to the solid state component, directly or indirectly, via covalent or non-covalent linkage, e.g., via a tag.
  • the tag can be any of a variety of components.
  • a molecule that binds the tag (a tag binder) is fixed to a solid support, and the tagged molecule of interest is attached to the solid support by interaction of the tag and the tag binder.
  • tags and tag binders can be used, based upon known molecular interactions well described in the literature.
  • a tag has a natural binder, for example, biotin, protein A, or protein G
  • tag binders avidin, streptavidin, neutravidin, the Fc region of an immunoglobulin, etc.
  • Antibodies to molecules with natural binders such as biotin are also widely available and appropriate tag binders (see, SIGMA Immunochemicals 1998 catalogue SIGMA, St. Louis MO).
  • any haptenic or antigenic compound can be used in combination with an appropriate antibody to form a tag/tag binder pair.
  • Thousands of specific antibodies are commercially available and many additional antibodies are described in the literature.
  • the tag is a first antibody and the tag binder is a second antibody which recognizes the first antibody.
  • receptor-ligand interactions are also appropriate as tag and tag-binder pairs, such as agonists and antagonists of cell membrane receptors (e.g., cell receptor-ligand interactions such as transferrin, c-kit, viral receptor ligands, cytokine receptors, chemokine receptors, interleukin receptors, immunoglobulin receptors and antibodies, the cadherin family, the integrin family, the selectin family, and the like; see, e.g., Pigott & Power, The Adhesion Molecule Facts Book I (1993 )).
  • cell membrane receptors e.g., cell receptor-ligand interactions such as transferrin, c-kit, viral receptor ligands, cytokine receptors, chemokine receptors, interleukin receptors, immunoglobulin receptors and antibodies, the cadherin family, the integrin family, the selectin family, and the like; see, e.g., Pigott & Power, The Adhesion Molecul
  • toxins and venoms can all interact with various cell receptors.
  • hormones e.g., opiates, steroids, etc.
  • intracellular receptors e.g., which mediate the effects of various small ligands, including steroids, thyroid hormone, retinoids and vitamin D; peptides
  • lectins e.g., which mediate the effects of various small ligands, including steroids, thyroid hormone, retinoids and vitamin D; peptides
  • drugs lectins
  • sugars e.g., nucleic acids (both linear and cyclic polymer configurations), oligosaccharides, proteins, phospholipids and antibodies
  • nucleic acids both linear and cyclic polymer configurations
  • oligosaccharides oligosaccharides
  • proteins e.g.
  • Synthetic polymers such as polyurethanes, polyesters, polycarbonates, polyureas, polyamides, polyethyleneimines, polyarylene sulfides, polysiloxanes, polyimides, and polyacetates can also form an appropriate tag or tag binder. Many other tag/tag binder pairs are also useful in assay systems described herein, as would be apparent to one of skill upon review of this disclosure.
  • linkers such as peptides, polyethers, and the like can also serve as tags, and include polypeptide sequences, such as poly-Gly sequences of between about 5 and 200 amino acids.
  • polypeptide sequences such as poly-Gly sequences of between about 5 and 200 amino acids.
  • Such flexible linkers are known to those of skill in the art.
  • poly(ethelyne glycol) linkers are available from Shearwater Polymers, Inc., Huntsville, Alabama. These linkers optionally have amide linkages, sulfhydryl linkages, or heterofunctional linkages.
  • Tag binders are fixed to solid substrates using any of a variety of methods currently available.
  • Solid substrates are commonly derivatized or functionalized by exposing all or a portion of the substrate to a chemical reagent which fixes a chemical group to the surface which is reactive with a portion of the tag binder.
  • groups which are suitable for attachment to a longer chain portion would include amines, hydroxyl, thiol, and carboxyl groups.
  • Aminoalkylsilanes and hydroxyalkylsilanes can be used to functionalize a variety of surfaces, such as glass surface. The construction of such solid phase biopolymer arrays is well described in the literature ( see, e.g., Merrifield, J. Am. Chem. Soc.
  • Non-chemical approaches for fixing tag binders to substrates include other common methods, such as heat, cross-linking by UV radiation, and the like.
  • the invention provides in vitro assays for identifying, in a high throughput format, compounds that can modulate the expression or activity of the polynucleotides or polypeptides of the invention.
  • the methods of the invention include such a control reaction.
  • "no modulator" control reactions that do not include a modulator provide a background level of binding activity.
  • a known activator of a polynucleotide or polypeptide of the invention can be incubated with one sample of the assay, and the resulting increase in signal resulting from an increased expression level or activity of polynucleotide or polypeptide determined according to the methods herein.
  • a known inhibitor of a polynucleotide or polypeptide of the invention can be added, and the resulting decrease in signal for the expression or activity can be similarly detected.
  • Yet another assay for compounds that modulate the activity of a polypeptide or polynucleotide of the invention involves computer assisted drug design, in which a computer system is used to generate a three-dimensional structure of the polypeptide or polynucleotide based on the structural information encoded by its amino acid or nucleotide sequence.
  • the input sequence interacts directly and actively with a pre-established algorithm in a computer program to yield secondary, tertiary, and quaternary structural models of the molecule.
  • Similar analyses can be performed on potential receptors or binding partners of the polypeptides or polynucleotides of the invention.
  • the models of the protein or nucleotide structure are then examined to identify regions of the structure that have the ability to bind, e.g., a polypeptide or polynucleotide of the invention. These regions are then used to identify polypeptides that bind to a polypeptide or polynucleotide of the invention.
  • the three-dimensional structural model of a protein is generated by entering protein amino acid sequences of at least 10 amino acid residues or corresponding nucleic acid sequences encoding a potential receptor into the computer system.
  • the amino acid sequences encoded by the nucleic acid sequences provided herein represent the primary sequences or subsequences of the proteins, which encode the structural information of the proteins.
  • At least 10 residues of an amino acid sequence (or a nucleotide sequence encoding 10 amino acids) are entered into the computer system from computer keyboards, computer readable substrates that include, but are not limited to, electronic storage media (e.g., magnetic diskettes, tapes, cartridges, and chips), optical media (e.g., CD ROM), information distributed by internet sites, and by RAM.
  • the three-dimensional structural model of the protein is then generated by the interaction of the amino acid sequence and the computer system, using software known to those of skill in the art.
  • the amino acid sequence represents a primary structure that encodes the information necessary to form the secondary, tertiary, and quaternary structure of the protein of interest.
  • the software looks at certain parameters encoded by the primary sequence to generate the structural model. These parameters are referred to as "energy terms,” and primarily include electrostatic potentials, hydrophobic potentials, solvent accessible surfaces, and hydrogen bonding. Secondary energy terms include van der Waals potentials. Biological molecules form the structures that minimize the energy terms in a cumulative fashion. The computer program is therefore using these terms encoded by the primary structure or amino acid sequence to create the secondary structural model.
  • the tertiary structure of the protein encoded by the secondary structure is then formed on the basis of the energy terms of the secondary structure.
  • the user at this point can enter additional variables such as whether the protein is membrane bound or soluble, its location in the body, and its cellular location, e.g., cytoplasmic, surface, or nuclear. These variables along with the energy terms of the secondary structure are used to form the model of the tertiary structure.
  • the computer program matches hydrophobic faces of secondary structure with like, and hydrophilic faces of secondary structure with like.
  • potential ligand binding regions are identified by the computer system.
  • Three-dimensional structures for potential ligands are generated by entering amino acid or nucleotide sequences or chemical formulas of compounds, as described above.
  • the three-dimensional structure of the potential ligand is then compared to that of a polypeptide or polynucleotide of the invention to identify binding sites of the polypeptide or polynucleotide of the invention. Binding affinity between the protein and ligands is determined using energy terms to determine which ligands have an enhanced probability of binding to the protein.
  • Computer systems are also used to screen for mutations, polymorphic variants, alleles and interspecies homologs of genes encoding a polypeptide or polynucleotide of the invention. Such mutations can be associated with disease states or genetic traits and can be used for diagnosis. As described above, GeneChipTM and related technology can also be used to screen for mutations, polymorphic variants, alleles and interspecies homologs. Once the variants are identified, diagnostic assays can be used to identify patients having such mutated genes.
  • Identification of the mutated a polypeptide or polynucleotide of the invention involves receiving input of a first amino acid sequence of a polypeptide of the invention (or of a first nucleic acid sequence encoding a polypeptide of the invention), e.g., any amino acid sequence having at least 60%, optionally at least 70% or 85%, identity with the amino acid sequence of interest, or conservatively modified versions thereof.
  • the sequence is entered into the computer system as described above.
  • the first nucleic acid or amino acid sequence is then compared to a second nucleic acid or amino acid sequence that has substantial identity to the first sequence.
  • the second sequence is entered into the computer system in the manner described above. Once the first and second sequences are compared, nucleotide or amino acid differences between the sequences are identified.
  • Such sequences can represent allelic differences in various polynucleotides of the invention, and mutations associated with disease states and genetic traits.
  • the invention provides compositions, kits and integrated systems for practicing the assays described herein using polypeptides or polynucleotides of the invention, antibodies specific for polypeptides or polynucleotides of the invention, etc.
  • the invention provides assay compositions for use in solid phase assays; such compositions can include, for example, one or more polynucleotides or polypeptides of the invention immobilized on a solid support, and a labeling reagent.
  • the assay compositions can also include additional reagents that are desirable for hybridization. Modulators of expression or activity of polynucleotides or polypeptides of the invention can also be included in the assay compositions.
  • kits for carrying out the therapeutic and diagnostic assays of the invention typically include a probe that comprises an antibody that specifically binds to polypeptides or polynucleotides of the invention, and a label for detecting the presence of the probe.
  • the kits may include several polynucleotide sequences encoding polypeptides of the invention.
  • Kits can include any of the compositions noted above, and optionally further include additional components such as instructions to practice a high-throughput method of assaying for an effect on expression of the genes encoding the polypeptides of the invention, or on activity of the polypeptides of the invention, one or more containers or compartments (e.g., to hold the probe, labels, or the like), a control modulator of the expression or activity of polypeptides of the invention, a robotic armature for mixing kit components or the like.
  • additional components such as instructions to practice a high-throughput method of assaying for an effect on expression of the genes encoding the polypeptides of the invention, or on activity of the polypeptides of the invention, one or more containers or compartments (e.g., to hold the probe, labels, or the like), a control modulator of the expression or activity of polypeptides of the invention, a robotic armature for mixing kit components or the like.
  • the invention also provides integrated systems for high-throughput screening of potential modulators for an effect on the expression or activity of the polypeptides of the invention.
  • the systems typically include a robotic armature which transfers fluid from a source to a destination, a controller which controls the robotic armature, a label detector, a data storage unit which records label detection, and an assay component such as a microtiter dish comprising a well having a reaction mixture or a substrate comprising a fixed nucleic acid or immobilization moiety.
  • a number of robotic fluid transfer systems are available, or can easily be made from existing components.
  • a Zymate XP Zymark Corporation; Hopkinton, MA
  • a Microlab 2200 Hamilton; Reno, NV
  • pipetting station can be used to transfer parallel samples to 96 well microtiter plates to set up several parallel simultaneous STAT binding assays.
  • Optical images viewed (and, optionally, recorded) by a camera or other recording device are optionally further processed in any of the embodiments herein, e.g., by digitizing the image and storing and analyzing the image on a computer.
  • a variety of commercially available peripheral equipment and software is available for digitizing, storing and analyzing a digitized video or digitized optical image, e.g., using PC (Intel x86 or Pentium chip-compatible DOS ® , OS2 ® WINDOWS ® , WINDOWS NT ® , WINDOWS95 ® , WINDOWS98 ® , or WINDOWS2000 ® based computers), MACINTOSH ® , or UNIX ® based (e.g., SUN ® work station) computers.
  • PC Intel x86 or Pentium chip-compatible DOS ® , OS2 ® WINDOWS ® , WINDOWS NT ® , WINDOWS95 ® , WINDOWS98 ® , or WINDOWS2000 ® based computers
  • MACINTOSH ® or UNIX ® based (e.g., SUN ® work station) computers.
  • One conventional system carries light from the specimen field to a cooled charge-coupled device (CCD) camera, in common use in the art.
  • a CCD camera includes an array of picture elements (pixels). The light from the specimen is imaged on the CCD. Particular pixels corresponding to regions of the specimen (e.g., individual hybridization sites on an array of biological polymers) are sampled to obtain light intensity readings for each position. Multiple pixels are processed in parallel to increase speed.
  • the apparatus and methods of the invention are easily used for viewing any sample, e.g., by fluorescent or dark field microscopic techniques.
  • Modulators of the polynucleotides or polypeptides of the invention can be administered directly to a mammalian subject for modulation of activity of those molecules in vivo. Administration is by any of the routes normally used for introducing a modulator compound into ultimate contact with the tissue to be treated and is well known to those of skill in the art. Although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route.
  • modulators of polynucleotides or polypeptides of the invention can be combined with other drugs useful for treating mental disorders including useful for treating mood disorders, e.g., schizophrenia, bipolar disorders, or major depression.
  • pharmaceutical compositions of the invention comprise a modulator of a polypeptide of polynucleotide of the invention combined with at least one of the compounds useful for treating schizophrenia, bipolar disorder, or major depression, e.g., such as those described in U.S. Patent Nos. 6,297,262 ; 6,284,760 ; 6,284,771 ; 6,232,326 ; 6,187,752 ; 6,117,890 ; 6,239,162 or 6,166,008 .
  • compositions of the invention may comprise a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions of the present invention ( see, e.g., Remington's Pharmaceutical Sciences, 17th ed. 1985 )).
  • the modulators e.g., agonists or antagonists
  • aerosol formulations i.e., they can be "nebulized" to be administered via inhalation or in compositions useful for injection.
  • Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.
  • Formulations suitable for administration include aqueous and non-aqueous solutions, isotonic sterile solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
  • compositions can be administered, for example, orally, nasally, topically, intravenously, intraperitoneally, or intrathecally.
  • the formulations of compounds can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials. Solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.
  • the modulators can also be administered as part of a prepared food or drug.
  • the dose administered to a patient should be sufficient to effect a beneficial response in the subject over time.
  • the optimal dose level for any patient will depend on a variety of factors including the efficacy of the specific modulator employed, the age, body weight, physical activity, and diet of the patient, on a possible combination with other drugs, and on the severity of the mental disorder.
  • the size of the dose also will be determined by the existence, nature, and extent of any adverse side effects that accompany the administration of a particular compound or vector in a particular subject.
  • a physician may evaluate circulating plasma levels of the modulator, modulator toxicity, and the production of anti-modulator antibodies.
  • the dose equivalent of a modulator is from about 1 ng/kg to 10 mg/kg for a typical subject.
  • modulators of the present invention can be administered at a rate determined by the LD-50 of the modulator, and the side effects of the modulator at various concentrations, as applied to the mass and overall health of the subject. Administration can be accomplished via single or divided doses.
  • a variety of human diseases can be treated by therapeutic approaches that involve stably introducing a gene into a human cell such that the gene is transcribed and the gene product is produced in the cell.
  • Diseases amenable to treatment by this approach include inherited diseases, including those in which the defect is in a single or multiple genes.
  • Gene therapy is also useful for treatment of acquired diseases and other conditions. For discussions on the application of gene therapy towards the treatment of genetic as well as acquired diseases, see, Miller, Nature 357:455-460 (1992 ); and Mulligan, Science 260:926-932 (1993 ).
  • gene therapy can be used for treating a variety of disorders and/or diseases in which the polynucleotides and polypeptides of the invention has been implicated.
  • compounds, including polynucleotides can be identified by the methods of the present invention as effective in treating a mental disorder.
  • Introduction by gene therapy of these polynucleotides can then be used to treat, e.g., mental disorders including mood disorders and psychotic disorders.
  • the polynucleotides of the invention can be incorporated into a vector.
  • vectors used for such purposes include expression plasmids capable of directing the expression of the nucleic acids in the target cell.
  • the vector is a viral vector system wherein the nucleic acids are incorporated into a viral genome that is capable of transfecting the target cell.
  • the polynucleotides can be operably linked to expression and control sequences that can direct expression of the gene in the desired target host cells. Thus, one can achieve expression of the nucleic acid under appropriate conditions in the target cell.
  • Viral vector systems useful in the expression of the nucleic acids include, for example, naturally occurring or recombinant viral vector systems.
  • suitable viral vectors include replication competent, replication deficient, and conditionally replicating viral vectors.
  • viral vectors can be derived from the genome of human or bovine adenoviruses, vaccinia virus, herpes virus, adeno-associated virus, minute virus of mice (MVM), HIV, Sindbis virus, and retroviruses (including but not limited to Rous sarcoma virus), and MoMLV.
  • the genes of interest are inserted into such vectors to allow packaging of the gene construct, typically with accompanying viral DNA, followed by infection of a sensitive host cell and expression of the gene of interest.
  • nucleic acids are conjugated to a cell receptor ligand for facilitated uptake (e.g., invagination of coated pits and internalization of the endosome) through an appropriate linking moiety, such as a DNA linking moiety ( Wu et al., J. Biol. Chem. 263:14621-14624 (1988 ); WO 92/06180 ).
  • nucleic acids can be linked through a polylysine moiety to asialo-oromucocid, which is a ligand for the asialoglycoprotein receptor of hepatocytes.
  • viral envelopes used for packaging gene constructs that include the nucleic acids of the invention can be modified by the addition of receptor ligands or antibodies specific for a receptor to permit receptor-mediated endocytosis into specific cells ( see, e.g., WO 93/20221 , WO 93/14188 , and WO 94/06923 ).
  • the DNA constructs of the invention are linked to viral proteins, such as adenovirus particles, to facilitate endocytosis ( Curiel et al., Proc. Natl. Acad. Sci. U.S.A. 88:8850-8854 (1991 )).
  • molecular conjugates of the instant invention can include microtubule inhibitors ( WO/9406922 ), synthetic peptides mimicking influenza virus hemagglutinin ( Plank et al., J. Biol. Chem. 269:12918-12924 (1994 )), and nuclear localization signals such as SV40 T antigen ( WO93/19768 ).
  • Retroviral vectors are also useful for introducing the nucleic acids of the invention into target cells or organisms.
  • Retroviral vectors are produced by genetically manipulating retroviruses.
  • the viral genome of retroviruses is RNA.
  • this genomic RNA is reverse transcribed into a DNA copy which is integrated into the chromosomal DNA of transduced cells with a high degree of stability and efficiency.
  • the integrated DNA copy is referred to as a provirus and is inherited by daughter cells as is any other gene.
  • the wild type retroviral genome and the proviral DNA have three genes: the gag, the pol and the env genes, which are flanked by two long terminal repeat (LTR) sequences.
  • LTR long terminal repeat
  • the gag gene encodes the internal structural (nucleocapsid) proteins; the pol gene encodes the RNA directed DNA polymerase (reverse transcriptase); and the env gene encodes viral envelope glycoproteins.
  • the 5' and 3' LTRs serve to promote transcription and polyadenylation of virion RNAs.
  • Adjacent to the 5' LTR are sequences necessary for reverse transcription of the genome (the tRNA primer binding site) and for efficient encapsulation of viral RNA into particles (the Psi site) ( see, Mulligan, In: Experimental Manipulation of Gene Expression, Inouye (ed), 155-173 (1983 ); Mann et al., Cell 33:153-159 (1983 ); Cone and Mulligan, Proceedings of the National Academy of Sciences, U.S.A., 81:6349-6353 (1984 )).
  • retroviral vectors The design of retroviral vectors is well known to those of ordinary skill in the art. In brief, if the sequences necessary for encapsidation (or packaging of retroviral RNA into infectious virions) are missing from the viral genome, the result is a cis-acting defect which prevents encapsidation of genomic RNA. However, the resulting mutant is still capable of directing the synthesis of all virion proteins. Retroviral genomes from which these sequences have been deleted, as well as cell lines containing the mutant genome stably integrated into the chromosome are well known in the art and are used to construct retroviral vectors.
  • the retroviral vector particles are prepared by recombinantly inserting the desired nucleotide sequence into a retrovirus vector and packaging the vector with retroviral capsid proteins by use of a packaging cell line.
  • the resultant retroviral vector particle is incapable of replication in the host cell but is capable of integrating into the host cell genome as a proviral sequence containing the desired nucleotide sequence.
  • the patient is capable of producing, for example, a polypeptide or polynucleotide of the invention and thus restore the cells to a normal phenotype.
  • Packaging cell lines that are used to prepare the retroviral vector particles are typically recombinant mammalian tissue culture cell lines that produce the necessary viral structural proteins required for packaging, but which are incapable of producing infectious virions.
  • the defective retroviral vectors that are used lack these structural genes but encode the remaining proteins necessary for packaging.
  • To prepare a packaging cell line one can construct an infectious clone of a desired retrovirus in which the packaging site has been deleted. Cells comprising this construct will express all structural viral proteins, but the introduced DNA will be incapable of being packaged.
  • packaging cell lines can be produced by transforming a cell line with one or more expression plasmids encoding the appropriate core and envelope proteins. In these cells, the gag, pol, and env genes can be derived from the same or different retroviruses.
  • a number of packaging cell lines suitable for the present invention are also available in the prior art. Examples of these cell lines include Crip, GPE86, PA317 and PG13 ( see Miller et al., J. Virol. 65:2220-2224 (1991 )). Examples of other packaging cell lines are described in Cone and Mulligan Proceedings of the National Academy of Sciences, USA, 81:6349-6353 (1984 ); Danos and Mulligan Proceedings of the National Academy of Sciences, USA, 85:6460-6464 (1988 ); Eglitis et al. (1988), supra; and Miller (1990), supra.
  • Packaging cell lines capable of producing retroviral vector particles with chimeric envelope proteins may be used.
  • amphotropic or xenotropic envelope proteins such as those produced by PA317 and GPX packaging cell lines may be used to package the retroviral vectors.
  • an antisense polynucleotide is administered which hybridizes to a gene encoding a polypeptide of the invention.
  • the antisense polypeptide can be provided as an antisense oligonucleotide (see, e.g ., Murayama et al., Antisense Nucleic Acid Drug Dev. 7:109-114 (1997 )).
  • Genes encoding an antisense nucleic acid can also be provided; such genes can be introduced into cells by methods known to those of skill in the art.
  • an antisense nucleotide sequence in a viral vector such as, for example, in hepatitis B virus (see, e.g., Ji et al., J. Viral Hepat. 4:167-173 (1997 )), in adeno-associated virus ( see, e.g., Xiao et al., Brain Res. 756:76-83 (1997 )), or in other systems including, but not limited, to an HVJ (Sendai virus)-liposome gene delivery system (see, e.g., Kaneda et al., Ann. NY Acad. Sci.
  • a viral vector such as, for example, in hepatitis B virus (see, e.g., Ji et al., J. Viral Hepat. 4:167-173 (1997 )), in adeno-associated virus ( see, e.g., Xiao et al., Brain Res. 756:76-83 (1997 )),
  • a "peptide vector” see, e.g., Vidal et al., CR Acad. Sci III 32:279-287 (1997 )
  • a gene in an episomal or plasmid vector see, e.g., Cooper et al., Proc. Natl. Acad. Sci. U.S.A. 94:6450-6455 (1997 ), Yew et al. Hum Gene Ther. 8:575-584 (1997 )
  • a gene in a peptide-DNA aggregate see, e.g., Niidome et al., J. Biol. Chem.
  • Upregulated transcripts listed in the biomarker tables herein which are correlated with mental disorders may be targeted with one or more short interfering RNA (siRNA) sequences that hybridize to specific sequences in the target, as described above.
  • siRNA short interfering RNA
  • Targeting of certain brain transcripts with siRNA in vivo has been reported, for example, by Zhang et al., J. Gene. Med., 12:1039-45 (2003 ), who utilized monoclonal antibodies against the transferrin receptor to facilitate passage of liposome-encapsulated siRNA molecules through the blood brain barrier.
  • Targeted siRNAs represent useful therapeutic compounds for attenuating the over-expressed transcripts that are associated with disease states, e.g., MDD, BP, and other mental disorders.
  • conditional expression systems such as those typified by the tet-regulated systems and the RU-486 system, can be used (see, e.g., Gossen & Bujard, PNAS 89:5547 (1992 ); Oligino et al., Gene Ther. 5:491-496 (1998 ); Wang et al., Gene Ther. 4:432-441 (1997 ); Neering et al., Blood 88:1147-1155 (1996 ); and Rendahl et al., Nat. Biotechnol. 16:757-761 (1998 )). These systems impart small molecule control on the expression of the target gene(s) of interest.
  • stem cells engineered to express a transcript of interest can implanted into the brain.
  • the vectors used for gene therapy are formulated in a suitable buffer, which can be any pharmaceutically acceptable buffer, such as phosphate buffered saline or sodium phosphate/sodium sulfate, Tris buffer, glycine buffer, sterile water, and other buffers known to the ordinarily skilled artisan such as those described by Good et al. Biochemistry 5:467 (1966 ).
  • a suitable buffer such as phosphate buffered saline or sodium phosphate/sodium sulfate, Tris buffer, glycine buffer, sterile water, and other buffers known to the ordinarily skilled artisan such as those described by Good et al. Biochemistry 5:467 (1966 ).
  • compositions can additionally include a stabilizer, enhancer, or other pharmaceutically acceptable carriers or vehicles.
  • a pharmaceutically acceptable carrier can contain a physiologically acceptable compound that acts, for example, to stabilize the nucleic acids of the invention and any associated vector.
  • a physiologically acceptable compound can include, for example, carbohydrates, such as glucose, sucrose or dextrans; antioxidants, such as ascorbic acid or glutathione; chelating agents; low molecular weight proteins or other stabilizers or excipients.
  • Other physiologically acceptable compounds include wetting agents, emulsifying agents, dispersing agents, or preservatives, which are particularly useful for preventing the growth or action of microorganisms.
  • Various preservatives are well known and include, for example, phenol and ascorbic acid. Examples of carriers, stabilizers, or adjuvants can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, PA, 17th ed. (1985 ).
  • the formulations of the invention can be delivered to any tissue or organ using any delivery method known to the ordinarily skilled artisan.
  • the nucleic acids of the invention are formulated in mucosal, topical, and/or buccal formulations, particularly mucoadhesive gel and topical gel formulations.
  • Exemplary permeation enhancing compositions, polymer matrices, and mucoadhesive gel preparations for transdermal delivery are disclosed in U.S. Patent No. 5,346,701 .
  • the gene therapy formulations of the invention are typically administered to a cell.
  • the cell can be provided as part of a tissue, such as an epithelial membrane, or as an isolated cell, such as in tissue culture.
  • the cell can be provided in vivo, ex vivo, or in vitro.
  • the formulations can be introduced into the tissue of interest in vivo or ex vivo by a variety of methods.
  • the nucleic acids of the invention are introduced into cells by such methods as microinjection, calcium phosphate precipitation, liposome fusion, or biolistics.
  • the nucleic acids are taken up directly by the tissue of interest.
  • the nucleic acids of the invention are administered ex vivo to cells or tissues explanted from a patient, then returned to the patient.
  • ex vivo administration of therapeutic gene constructs include Nolta et al., Proc Natl. Acad. Sci. USA 93(6):2414-9 (1996 ); Koc et al., Seminars in Oncology 23 (1):46-65 (1996 ); Raper et al., Annals of Surgery 223(2):116-26 (1996 ); Dalesandro et al., J. Thorac. Cardi. Surg., 11(2):416-22 (1996 ); and Makarov et al., Proc. Natl. Acad. Sci. USA 93(1):402-6 (1996 ).
  • the present invention also provides methods of diagnosing mood disorders (such as major depression or bipolar disorder), psychotic disorders (such as schizophrenia), or a predisposition of at least some of the pathologies of such disorders. Diagnosis involves determining the level of a polypeptide or polynucleotide of the invention in a patient and then comparing the level to a baseline or range. Typically, the baseline value is representative of a polypeptide or polynucleotide of the invention in a healthy person not suffering from a mood disorder or a psychotic disorder or under the effects of medication or other drugs.
  • Variation of levels of a polypeptide or polynucleotide of the invention from the baseline range indicates that the patient has a mood disorder or a psychotic disorder or at risk of developing at least some aspects of a mood disorder or a psychotic disorder.
  • the level of a polypeptide or polynucleotide of the invention are measured by taking a blood, urine or tissue sample from a patient and measuring the amount of a polypeptide or polynucleotide of the invention in the sample using any number of detection methods, such as those discussed herein.
  • Antibodies can be used in assays to detect differential protein expression in patient samples, e.g., ELISA assays, immunoprecipitation assays, and immunohistochemical assays.
  • PCR assays can be used to detect expression levels of nucleic acids, as well as to discriminate between variants in genomic structure, such as insertion/deletion mutations (e.g., PSPHL).
  • the genomic structure of a gene such as PSPHL can be evaluated with known methods such as PCR to detect deletion or insertion mutations associated with disease suspectibility.
  • diagnosis can be made by detecting the presence or absence of mRNA or protein, or by examining the genomic structure of the gene.
  • Any combination of exons or non-transcribed regions can be used to detect the deletion allele. For example, the presence of exon 4 but not exons 1, 2, and/or 3 would indicate the presence of the deletion allele.
  • deletion of the promoter region would indicate the deletion allele. Any significant mRNA detection, especially detection of an mRNA comprising exons 1, 2, and/or 3, would indicate the absence of the deletion allele, which is not transcribed due to the promoter deletion.
  • Single nucleotide polymorphism (SNP) analysis is also useful for detecting differences between alleles of the polynucleotides (e.g., genes) of the invention.
  • SNPs linked to genes encoding polypeptides of the invention are useful, for instance, for diagnosis of diseases (e.g., mood disorders such as bipolar disease, major depression, and schizophrenia disorders) whose occurrence is linked to the gene sequences of the invention.
  • diseases e.g., mood disorders such as bipolar disease, major depression, and schizophrenia disorders
  • the individual is likely predisposed for one or more of those diseases.
  • the individual is homozygous for a disease-linked SNP, the individual is particularly predisposed for occurrence of that disease.
  • the SNP associated with the gene sequences of the invention is located within 300,000; 200,000; 100,000; 75,000; 50,000; or 10,000 base pairs from the gene sequence.
  • Various real-time PCR methods can be used to detect SNPs, including, e.g., Taqman or molecular beacon-based assays (e.g., U.S. Patent Nos. 5,210,015 ; 5,487,972 ; Tyagi et al., Nature Biotechnology 14:303 (1996 ); and PCT WO 95/13399 are useful to monitor for the presence of absence of a SNP.
  • Additional SNP detection methods include, e.g., DNA sequencing, sequencing by hybridization, dot blotting, oligonucleotide array (DNA Chip) hybridization analysis, or are described in, e.g., U.S. Patent No.
  • PCR methods can also be used to detect deletion/insertion polymorphisms, such as the deletion polymorphism of the PSPHL gene associated with suspectibility to BP.
  • the level of the enzymatic product of a polypeptide or polynucleotide of the invention is measured and compared to a baseline value of a healthy person or persons. Modulated levels of the product compared to the baseline indicates that the patient has a mood disorder or a psychotic disorder or is at risk of developing at least some aspects of a mood disorder or a psychotic disorder.
  • Patient samples for example, can be blood, urine or tissue samples.
  • Major depressive disorder and bipolar disorder (BP) are affective diseases that strike a significant proportion of the population. These complex genetic disorders arise from the interplay of vulnerability genes and environmental stressors, impacting neural circuits that control mood. Beyond the role of limbic structures, mood disorders are hypothesized to involve aberrant activity of the cerebral cortex.
  • imaging techniques have implicated the dorsolateral prefrontal (DLPFC) and anterior cingulate (AnCg) cortices in mood disorders since affected subjects display changes in volumetric measurements ( Harrison, P. J. Brain 125, 1428-49 (2002 ).) and altered activity in response to a cognitive challenge. ( Kruger, S., Seminowicz, D., Goldapple, K., Kennedy, S. H.
  • the Affymetrix HG-133A array contains probe sets for 21 FGF system transcripts, including all 4 receptors (FGFR1, 2, 3, 4) and 12 FGF peptide ligands (FGF1, 2, 3, 5, 6, 7, 8, 9, 12, 13, 14, 17, 18, 20, 21, 22, 23). Of these only 10 were reliably detected in the regions assayed and include three of the FGF receptors (FGFR1, 2, and 3) and seven FGF ligands (FGF1, 2, 7, 9, 12, 13,14).
  • BDNF brain derived neurotrophic factor
  • a smaller volume of literature implicates other growth factors, including nerve growth factor ( Parikh, V., Evans, D. R., Khan, M. M. & Mahadik, S. P.
  • FGF2 Viti, J., Gulacsi, A. & Lillien, L. J Neurosci 23, 5919-27 (2003 )
  • FGF8 Gunhaga, L. et al. Nat Neurosci 6, 701-7 (2003 )
  • FGF2 promotes neuronal survival and axonal branching ( Abe, K. & Saito, H. Pharmacol Res 43, 307-12 (2001 )) and its expression is modulated by stress ( Molteni, R. et al., Brain Res Rev 37, 249-58 (2001 )).
  • Example 2 Identification of novel insertion/deletion polymorphism in PSPHL gene and association of deletion mutation with BP susceptibility
  • PSPHL shows dichotomous present/absent pattern of expression among individuals with brain-wide consistency suggests genetic variation in its regulation. Since genomic organization of PSPHL has not been characterized (Planitzer, supra (1998)), we have identified genomic organization of PSPHL as shown in Figure 14 .
  • the PSPHL gene consists of 4 exons. Exons 1, 2, 3 and 4 are 213 bp, 114 bp, 122 bp and 501 bp, in length, respectively, and span introns 1, 2 and 3 (3221bp, 829 bp and 11939 bp, in length, respectively).
  • the PSPHL gene has two alternative transcripts, one of which utilizes the exons 1-4 (PSPHL-A in figure 14 ), while another utilizes the exons 1, 2 and 4 (PSPHL-B).
  • PSPHL-A in figure 14
  • PSPHL-B the exons 1, 2 and 4
  • the deleted genomic region spans more than 30 kb, including the promoter region and the exons 1, 2 and 3 of PSPHL gene. This genetic variance explains the present/absent pattern of the PSPHL expression.
  • An over-representation of the deletion allele resulting in the absence of PSPHL expression increases susceptibility to BPD.
  • PSPHL and PSPH are highly homologous, but appear to be different genes, which are about 200kb apart from each other on chromosome 7p11.2 region. Especially, exons 2-4 of PSPHL are highly homologous to exons 4 and 8 of PSPH gene.
  • Predicted amino acid sequences of PSPH, PSPHL-A and PSPHL-B are shown in Figure 15 .
  • PSPHL-A and PSPHL-B share N-terminal 57 common amino acids, transcribed from exons 1 and 2.
  • PSPHL-A has unique C-terminal 36 amino acids, transcribed from exon3, while PSPHL-B has unique C-terminal 17 amino acids, transcribed from exon 4.
  • PSPH and PSPHL-A&B have 31 amino acids in common.
  • the common amino acids locates at the N-terminal end of PSPH and middle region (25th - 56th amino acids) of PSPHL-A and B.
  • the common region contains consensus phosphorylation site of Na/K ATPase and casein kinase II phospholyration site. Based on the similarity in the structure, PSPHL shares some function with PSPH gene.
  • PSPH is the rate limiting enzyme for serine synthesis.
  • PSPH has haloacid dehalogenase-like hydrolase domain, which is responsible for the activity. Greater than 90% of L-serine in brain is formed via the phosphorylated pathway.
  • PSPH may be dimeric from of the enzyme with a monomeric molecular weight of 26kDa.
  • L-serine is converted to sphingomyelins and gangliosides, as well as L-glycine and D-serine, both of which act as coagonist for NMDA receptor associated glycine binding site.
  • L-glycine is also an agonist for strychnine-sensitive glycine receptor ( Figure 16 ).
  • PSPHL is involved in serine amino acid metabolic pathway, and may involved in other pathways as well.
  • FGF-2 injected rats exhibited a 10.5% increase in dentate gyrus volume.
  • the results show that FGF-2 significantly increased locomotor activity over controls in a novel environment. Increased activity in response to novelty has been associated with a host of other measures including decreased anxiety-like behavior.
  • adult rats that received FGF-2 as neonates also performed significantly better than controls in the Morris water maze.
  • Cohort A consisted of 22 subjects including 7 healthy control subjects, 6 patients with BPD and 9 patients with MDD.
  • agonal conditions including hypoxia, coma, pyrexia, seizure, dehydration, hypoglycemia, multi-organ failure, skull fracture, ingestion of neurotoxic substances or prolonged agonal duration, which is known to affect tissue pH, RNA integrity and gene expression profile in postmortem brain, and showed brain tissue pH of more than 6.5.
  • Experiment 2 For further technical replication, samples from the 22 subjects from cohort A were reanalyzed in AnCg and DLPFC utilizing U133A GeneChips at two laboratories.
  • Experiment 3 Samples from the additional cohort B were analyzed on U133A GeneChips in AnCg and DLPFC at two laboratories. Signal intensity data was extracted with Robust Multi-array Average (RMA) for each probe set and each subject. Gene-wise Pearson's correlation coefficients between experimental duplicates were calculated, and only the genes significantly correlated between experimental duplicates were considered to be reliably detectable genes, and subjected to the downstream analyses. For these reliably detectable genes, mixed-model multivariate ANOVA analyses were employed utilizing Partek Pro 6.0 (Partek, St.Charles, MO) to adjust the effect of the diagnostic classification (BPD, MDD, control) for possible confounders, including site for experiment, experimental batch, and gender.
  • RMA Robust Multi-array Average
  • FIG 26A and Table 14 summarize cAMP signaling pathway related genes which were differentially expressed in anterior cingulate cortex (AnCg) of bipolar disorder (BPD) patients compared with controls.
  • AnCg anterior cingulate cortex
  • BPD bipolar disorder
  • NPY1 G protein inhibitory subunit 1
  • GPM3 G protein inhibitory subunit 3
  • Somatostatin (SST) a ligand for Gi-coupled GPCR, was significantly increased in AnCg in our microarray data.
  • GNAI1 G protein alpha subunit inhibitory peptide 1
  • PDE1A phosphodiesterase 1A
  • PKIA Protein kinase A inhibitor alpha
  • CDK5 cyclin dependent kinase 5
  • PDE8A protein phosphatase 1, catalytic subunit, alpha
  • PPP1CA protein phosphatase 1, catalytic subunit, alpha
  • mRNA expression of molecules suppressing cAMP concentration and PKA activity were generally increased in BPD, while molecules activating cAMP signaling (Gs-coupled GPCR, Gs, adenylate cyclase, protein kinase A) did not show significant alteration at the transcript level.
  • FIG. 26 and Table 14 summarize cAMP signaling pathway related genes which were differentially expressed in AnCg of MDD patients compared with controls.
  • Gi-linked endothelial differentiation GPCR 1 EDG1
  • Regulator of G protein signaling 20 RGS20
  • PDE8A phosphodiesterase 8A
  • PPP1R3C protein phosphatase 1 regulatory subunit 3C
  • Expression levels of PDE8A and PPP1R3C mRNAs were significantly lower also in DLPFC of MDD (Table 15).
  • Significant decrease in RGS20 expression in MDD was observed also in CB (Table 16).
  • mRNA expression of molecules suppressing cAMP concentration and PKA activity were generally decreased in MDD, while the molecules activating cAMP signaling did not show significant alteration at the transcript level.
  • FIG. 26C and Table 14 summarize phosphatidylinositol signaling (PI) pathway related genes which were differentially expressed in AnCg of BPD patients compared with controls.
  • PI phosphatidylinositol signaling
  • IPP1 inositol polyphosphate-1-phosphatase
  • CDS1 CDP-diacylglycerol synthase 1
  • PIK3R1 regulatory subunit of class I phosphatidylinositol 3 kinase
  • PKCI protein kinase C iota
  • Inositol 1, 4, 5-trisphosphate 3-kinase B (ITPKB) and catalytic beta subunit of class II phosphatidylinositol 3 kinase (PIK3C2B) did not reach significance criteria, but increased by 10%-20% in AnCg of BPD.
  • Figure 26D and Table 14 summarize PI signaling pathway related genes which were differentially expressed in AnCg of MDD patients compared with controls.
  • Gq-linked neurotensin receptor 2 (NTSR2) and endothelin receptor type B (EDNRB) were significantly decreased in AnCg of MDD.
  • Messenger RNA expression of inositol polyphosphate-5-phosphatase F (INPP5F) was significantly higher, while inositol 1, 4, 5-trisphosphate 3-kinase B (ITPKB) and catalytic alpha subunit of class II phosphatidylinositol 3 kinase (PIK3C2A) were significantly lower in AnCg of MDD compared with control.
  • IPP5F inositol polyphosphate-5-phosphatase F
  • IPKB 5-trisphosphate 3-kinase B
  • PIK3C2A catalytic alpha subunit of class II phosphatidylinositol 3 kina
  • Inositol polyphosphate-5-phosphatase A Inositol polyphosphate-5-phosphatase A
  • PLCB 1 protein kinase C beta 1
  • IPR1 5-triphosphate receptor type 1
  • G protein-coupled receptor family C group 5, member B (GPRC5B) and G protein-coupled receptor 37 (GPR37).
  • GPRC5B was significantly increased in AnCg and DLPFC of BPD.
  • GPRC5B was significantly decreased in AnCg, DLPFC and CB of MDD patients.
  • a significant decrease of GPRC5B in AnCg and DLPFC of MDD patients was replicated by the experiments utilizing another independent cohort B.
  • GPR37 was also significantly increased in AnCg of BPD, and significantly decreased in AnCg, DLPFC and CB of MDD.
  • mRNA expression levels by real-time quantitative reverse transcriptase PCR for the following 7 genes, in anterior cingulate cortex (AnCg): Somatostatin (SST), neuropeptide Y (NPY), G protein-coupled receptor C-5-B (GPRC5B), G protein-coupled receptor 37 (GPR37), regulator of G-protein signaling 20 (RGS20), inositol polyphosphate-1-phosphatase (INPP1) and protein phosphatase 1 regulatory subunit 3C(PPP1R3C).
  • SST Somatostatin
  • NPY neuropeptide Y
  • GPR37 G protein-coupled receptor 37
  • RAS20 regulator of G-protein signaling 20
  • IPP1 inositol polyphosphate-1-phosphatase
  • PPP1R3C protein phosphatase 1 regulatory subunit 3C
  • qRT-PCR data showed that mRNA expressions of neuropeptide Y (NPY), G protein-coupled receptor C-5-B (GPRC5B), G protein-coupled receptor 37 (GPR37), inositol polyphosphate-1-phosphatase (INPP1) were significantly increased in AnCg of BPD, and expression level of GPRCSB, GPR37, regulator of G-protein signaling 20 (RGS20) and protein phosphatase 1 regulatory subunit 3C (PPP1R3C) were significantly decreased in the AnCg of MDD group. While somatostatin (SST) mRNA expression was increased in AnCg of BPD in both microarray experimental duplicates utilizing U95Av2 and U133A, the finding was not replicated by qRT-PCR (Table 17).
  • GPR37 mRNA is preferentially expressed in subcortical white matter. GPR37 expression in the deeper layers (V-VI) is relatively higher than the superficial layers (I-III). GPR37 mRNA expression in subcortical white matter was higher in AnCg of the BPD subjects compared to the control subjects. GPR37 mRNA expression was rarely detected in AnCg of the MDD subjects analyzed.
  • Figure 27 shows the dysregulation of genes involved in cAMP- and phosphatidylinositol signaling pathways in brain tissue from patients with BPD and MDD.
  • Table 18 summarizes genes which are differentially expressed in amygdala, hippocampus and nucleus accumbens of BPD patients.
  • Table 19 summarizes genes differentially expressed in the three brain regions of MDD.
  • This Example shows gene dysregulation in pathways related to Mitochondria, Proteasome, Apoptosis, and Chaperone in Mood Disorder.
  • Three brain regions were studied: AnCg, Cerebellum, and DLPFC. The results are compiled in Tables 20-22.
  • NCAM1 ASSOCIATION WITH BIPOLAR DISORDER AND SCHIZOPHRENIA AND SPLICE VARIANTS OF NCAM1.
  • Genomic DNA was extracted from human postmortem brain cerebellum tissue. Primers were designed for SNP 9 and then tested via PCR to determine correct band size. Using the SNP 9 primers, gDNA of 40 cases (20 controls, 9 BPDs and 11 MDDs) was sequenced with both the forward and reverse primers. The SNPs were located in exons a, b and c. SNPs b and c are intronic and found just before exon 'b' (7 bps upstream) and 'c' (12 bps upstream) respectively. Exon 'a' did not have a SNP in close proximity.
  • the genotypes were collected on an additional 26 bipolar genomic DNA samples extracted from lymphocytes from the National Institute of Mental Health (NIMH) for all 4 SNPs: SNP 6, SNP 9, SNP b and SNP c (see Figure 26 ).
  • the Stanley samples were genotyped for SNP 9 and SNP b.
  • the three groups of bipolar cases were combined and three control groups were merged and used for statistical comparisons of SNP 9 and SNP b.
  • the results show an association of SNP 9 and SNP b haplotype with bipolar disorder and schizophrenia.
  • the splice variants are alternative splicing combinations of 3 mini-exons (a,b,c) with the fourth SEC exon are shown in Figure 30 .
  • Two mood disorders were tested (Bipolar Disorder, Type I and Major Depressive Disorder, Recurrent) and both showed differences in NCAM1 splice variants in the DLPFC.
  • the present data relates NCAM1 polymorphic variation to bipolar disorder and splice variations in mRNA occurring near the polymorphisms.
  • a genotypic association between SNP b in NCAM1 and bipolar disorder and a suggestive association of SNP 9 with schizophrenia were found.
  • Three of the two marker haplotypes for SNP 9 and SNP b, CT, C(T/C), and (C/A)(T/C) display varying frequency distribution between bipolar and controls.
  • Schizophrenia and controls show differences in frequency distribution in four of the two marker haplotypes of SNP 9 and SNP b, CT, C(T/C), (C/A)(T/C) and (C/A)C.
  • Bipolar disorder differs from schizophrenia for SNP 9 and SNP b by haplotype frequency differences.
  • SNP b and SNP 9 are not in LD and they are individually related to schizophrenia (SNP 9) and bipolar disorder (SNP b).
  • the splice variant evidence for SNP 9 and b confirm that each SNP can be associated with differences in SEC exon splicing, thus providing some differential mechanisms for release of NCAM1 in the brain.
  • This finding concerning the difference in splice variant relative amounts as a function of certain genotypes was shown in three of the four SNPs where at least one genotype showed a difference in the amount of SEC by splice variant. This evidence suggests that the amount of SEC in brain is not regulated by just one genotype.
  • SNP 9 and SNP b are significantly different between controls and bipolar and between controls and schizophrenia this may support the observation of differential splicing patterns of the SEC exon found across many samples. Additionally, SNP 9 and SNP b are not in LD and thus the individual associations in schizophrenia and bipolar with these SNPs also may be transmitted through differential splicing patterns. The SEC exon was clearly regulated by certain combinations of mini-exons. We have identified discrete splice variants that can be further studied and are perhaps associated with regulatory intronic SNPs.
  • Lithium has long been the drug of choice for treating manic-depressive illness (manic-depression; bipolar affective disorder, BPD).
  • This Example shows non-human primate genes which exhibit differential expression in response to treatment with lithium. The results have implications for understanding the mood stabilizing effects of lithium in patients with manic depression.
  • Gene expression profiling was carried out on the anterior cingulate cortex (AnCg) using high-density oligonucleotide microarrays (Affymetrix GeneChips).
  • Affymetrix GeneChips high-density oligonucleotide microarrays
  • MDD Major depressive disorder
  • BPD bipolar affective disorder
  • mania emotional highs
  • BPD bipolar affective disorder
  • Li 2 CO 3 lithium carbonate
  • Lithium unlike other anti-manic treatment agents is unique in its ability to abort the manic condition and restore patient's balanced mental status.
  • AnCg showed a total of 220 candidate transcripts (65 upregulated and 155 down-regulated).
  • the candidate genes from AnCg are listed in Table 28.
  • Ontological annotations mapped candidate genes to several different biological processes and pathways, including GSK3B signaling system, as predicted, in the AnCg.
  • V-ATPase subunits Of the 14 V-ATPase subunits that we have interrogated with Affy microarrays and Illumina microarrays, 7 subunits (50%) are differentially expressed (P ⁇ 0.05) in hippocampal MDD versus control on either the Affymetrix or Illumina arrays. Three of the 7 subunits are differentially expressed in MDD hippocampus on both the Affymetrix and Illumina arrays (see Table 29). Two of the V-ATPase subunits are also differentially expressed (P ⁇ 0.05) in our Affy microarray study of monkey hippocampus (i.e., Table 30, showing chronic social stress versus no-stress comparison). These findings demonstrate that drugs now being developed to inhibit V-ATPase in patients with cancer and osteoporosis may also prove useful as novel antidepressants.

Abstract

The present invention provides methods for diagnosing mental disorders. The invention also provides methods of identifying modulators of mental disorders as well as methods of using these modulators to treat patients suffering from mental disorders.

Description

    CROSS-REFERENCES TO RELATED APPLICATIONS
  • The present application is related to USSN 60/581,998, filed June 21, 2004, and USSN 60/621,252, filed October 22, 2004, and USSN 60/667,296, filed March 31, 2005 each of which is incorporated herein in its entirety by reference.
  • STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT
  • Not applicable.
  • BACKGROUND OF THE INVENTION
  • Clinical depression, including both bipolar disorders and major depression disorders, is a major public health problem, affecting an estimated 9.5% of the adult population of the United States each year. While it has been hypothesized that mental illness, including mood disorders such as major depression ("MDD") and bipolar disorder ("BP") as well as psychotic disorders such as schizophrenia, may have genetic roots, little progress has been made in identifying gene sequences and gene products that play a role in causing these disorders, as is true for many diseases with a complex genetic origin (see, e.g., Burmeister, Biol. Psychiatry 45:522-532 (1999)).
  • The current lack of biomarkers and the ineffectiveness and reliability of the diagnosis and rates are important issues for the treatment of mental disorders. For example, around 15% of the population suffers from MDD while approximately 1% suffers from BP disorders. Diagnosing bipolar disorder is difficult when, as sometimes occurs, the patient presents only symptoms of depression to the clinician. At least 10-15% of BP patients are reported to be misdiagnosed as MDD. The consequences of such misdiagnosis include a delay in being introduced to efficacious treatment with mood stabilizers and a delay in seeking or obtaining counseling specific to bipolar disorder. Also treatment with antidepressants alone induces rapid cycling, switching to manic or mixed state, and consequently increases suicide risk. Furthermore, in addition to a lack of efficacy, long onset of action and side effects (sexual, sleep, weight gain, etc.), there are recent concerns relating to the undesirable effects of ADs on metabolic syndromes, such as diabetes and hypercholesteremia.
  • BRIEF SUMMARY OF THE INVENTION
  • Relying on the discovery that certain genes expressed in particular brain pathways and regions are likely involved in the development of mental illness, the present invention provides methods for diagnosis and treatment of mental illness, as well as methods for identifying compounds effective in treating mental illness.
  • In order to further understand the neurobiology of mood disorders such as bipolar disorders (BP) and major depression disorders (MDD), the inventors of the present application have used DNA microarrays to study expression profiles of human post-mortem brains from patients diagnosed with BP or MDD. In one aspect, the present invention relates to differential gene expression in the Anterior Cingulate (AnCg), Dorsolateral Prefrontal (DLPFC), and Cerebellar (CB) cortices, Hippocampus, Nucleus Accumbens and Amygdala regions of the brain, wherein the differential gene expression is associated with Bipolar Disorder (BPD) and Major Depressive Disorder (MDD). Certain genes in these regions are considered "unique" to a given disorder such as BP or MDD when differentially expressed in a particular mood disorder and not another (see, e.g., Tables 3, 4, 14-20). In other cases, where the genes are differentially expressed in both BP and MDD relative to healthy controls, the genes are considered to be involved in both disorders. Expression of the differentially expressed genes may be detected using any suitable methods of detection, e.g., microarrays, PCR or in situ hybridization. Gene expression may be detected in brain tissue, brain tissue samples, or other tissue samples (e.g., blood samples in the case of NCAM1).
  • In one aspect, the present invention relates to differential gene expression associated with G protein-coupled receptors (GPCR)s and downstream signaling pathways, mediated by cyclic adenosine monophosphate (cAMP) and phosphatidylinositol (PI), wherein the gene expression differentially occurs in the Anterior Cingulate (AnCg), Dorsolateral Prefrontal (DLPFC), and Cerebellar (CB) cortices, Hippocampus, Nucleus Accumbens and Amygdala regions of the brains of patients with Bipolar Disorder (BPD) and/or Major Depressive Disorder (MDD), relative to healthy controls (see, e.g., Tables 14-20).
  • In another aspect, the present invention relates to differential gene expression associated with G protein-coupled receptors (GPCR)s and downstream signaling pathways, mediated by cyclic adenosine monophosphate (cAMP) and phosphatidylinositol (PI), wherein the gene expression differentially occurs in the Hippocampus, Nucleus Accumbens and Amygdala regions of the brains of patients with Bipolar Disorder (BPD) and/or Major Depressive Disorder (MDD), relative to healthy controls (see, e.g., Tables 18-20).
  • The present invention also demonstrates differential expression of the FGF pathway in the frontal cortex of MDD subjects. Particular FGF-related genes, such as FGF2, are dysregulated by antidepressant therapy, environmental complexity, and the correlation to anxiety-like behavior (see, e.g., Tables 1a, 1b, and 2, and Figures 1-7 and 22). The FGF pathway is also related to neurogenesis, e.g., neural stem cell proliferation and differentiation, and the genes disclosed herein can be used for diagnosis and therapeutics related to neurogenesis. Furthermore, Figure 23 shows the effects of postnatal FGF-2 adminstration on neurogenesis, emotionality and gene expression in adult rats. The FGF injected animals exhibit significantly increased cell survival and proliteration in the dentate gyrus of the hippocampus. As adults, the animals show higher locomotor activity in a novel environment, an index of lower anxiety, and have better learning and memory.
  • The present invention also demonstrates that the genes of the glutamate/GABA signaling pathways are involved in MDD and BP (see Figure 24 and Table 8).
  • The present invention also demonstrates that mitrochondrial genes are involved in MDD andBP (see Table 10).
  • The present invention also demonstrates that 40 genes encoding growth factor family members and growth factor receptors are significantly differentially expressed in BP or MDD in the DLPFC or AnCg (see Tables 5, 6 and 7).
  • The present invention demonstrates that genes involved in G protein coupled receptors and their downstream signaling pathways , including cyclic AMP, phosphatidylinositol, and mitogen-activated protein kinase signaling pathways are dysregulated in BP and/or MDD (see Tables 6 and 9 and Figures 8-13 and 17-19).
  • Finally the present invention provides for the first time a novel insertion/deletion polymporphism in the phosphoserine phosphatase-like gene (PSPHL) and demonstrates that a novel deletion polymorphism of PSPHL is related to susceptibility to bipolar disorder. Therefore, detection of this polymorphism is useful for diagnosis of BP, as well as for drug discovery assays for BP therapeutics. In addition, the serine amino acid metabolic pathway, of which PSPHL is a member, is a target for drug discovery for BP therapeutics. The PSPHL gene was first cloned by Planitzer et al., Gene 210 297-306 (1998). The accession number for a representative nucleic acid sequence is AJ0016112 and the accession number for a representative protein sequence is CAA04865.1. See Figures 14-16.
  • The present invention demonstrates, for the first time, unique expression of the 24 nucleic acids listed in Table 3 in the brains of bipolar disorder subjects but not major depression subjects; the unique expression of the 24 nucleic acids listed in Table 4 in the brains of major depression subjects but not bipolar subjects, and the differential and/or unique expression of the nucleic acids listed in Tables 5-10 in the brains of patients suffering from bipolar disorder and major depression disorder, in comparison with normal control subjects. In addition, the present invention identifies biochemical pathways involved in uniquely or differentially in mood disorders, where the proteins encoded by the nucleic acids listed in Table 3-10 are components of the biochemical pathways (e.g., the growth factor, e.g., FGF, signal transduction pathway, GPCR signal transduction pathways, mitochondrial pathways, and glutamate/GABA signaling pathways). Furthermore, the invention demonstrates the unique expression of a PSPHL deletion polymorphism and it's associate with BP.
  • Genes and pathways that are uniquely or differentially expressed in MDD or BP are useful in diagnosing mood disorders and in assaying for therapeutics that can specifically treat MDD or BP, or can be used to treat both MDD and BP. Differential expression by brain region similarly is a useful diagnostic and therapeutic tool, as certain mood disorders primarily affect certain brain regions. Each brain region plays a unique and critical role in the overall phenotype of any particular mood disorder. Furthermore, because of the relationship between BP and psychotic disorders such as schizo affective disorders, the gene described herein unique to BP can also be uniquely expressed in schizophrenia, and so can be used for differential diagnosis with MDD.
  • This invention thus provides methods for determining whether a subject has or is predisposed for a mental disorder such as bipolar disorder or major depression disorder. The invention also provides methods of providing a prognosis and for monitoring disease progression and treatment. Furthermore, the present invention provides nucleic acid and protein targets for assays for drugs for the treatment of mental disorders such as bipolar disorder and major depression disorder.
  • In some embodiments, the methods comprise the steps of: (i) obtaining a biological sample from a subject; (ii) contacting the sample with a reagent that selectively associates with a polynucleotide or polypeptide encoded by a nucleic acid that hybridizes under stringent conditions to a nucleotide sequence listed in Tables 3-10 and Figure 14; and (iii) detecting the level of reagent that selectively associates with the sample, thereby determining whether the subject has or is predisposed for a mental disorder.
  • In some embodiments, the reagent is an antibody. In some embodiments, the reagent is a nucleic acid. In some embodiments, the reagent associates with a polynucleotide. In some embodiments, the reagent associates with a polypeptide. In some embodiments, the polynucleotide comprises a nucleotide sequence listed in Table 3-6. In some embodiment, the polypeptide comprises an amino acid sequence of a gene listed in Table 3-6. In some embodiments, the level of reagent that associates with the sample is different (i.e., higher or lower) from a level associated with humans without a mental disorder. In some embodiments, the biological sample is obtained from amniotic fluid. In some embodiments, the mental disorder is a mood disorder. In some embodiments, the mood disorder is selected from the group consisting of bipolar disorder and major depression disorder.
  • The invention also provides methods of identifying a compound for treatment of a mental disorder. In some embodiments, the methods comprises the steps of: (i) contacting the compound with a polypeptide, which is encoded by a polynucleotide that hybridizes under stringent conditions to a nucleic acid comprising a nucleotide sequence of Table 2 , 3, or 4; and (ii) determining the functional effect of the compound upon the polypeptide, thereby identifying a compound for treatment of a mental disorder.
  • In some embodiments, the contacting step is performed in vitro. In some embodiment, the polypeptide comprises an amino acid sequence of a gene listed in Table 3-6. In some embodiments, the polypeptide is expressed in a cell or biological sample, and the cell or biological sample is contacted with the compound. In some embodiments, the mental disorder is a mood disorder or psychotic disorder. In some embodiments, the mood disorder is selected from the group consisting of bipolar disorder and major depression. In some embodiments, the psychotic disorder is schizophrenia. In some embodiments, the methods further comprise administering the compound to an animal and determining the effect on the animal, e.g., an invertebrate, a vertebrate, or a mammal. In some embodiments, the determining step comprises testing the animal's mental function.
  • In some embodiments, the methods comprise the steps of (i) contacting the compound to a cell, the cell comprising a polynucleotide that hybridizes under stringent conditions to a nucleotide sequence of Table 3-6; and (ii) selecting a compound that modulates expression of the polynucleotide, thereby identifying a compound for treatment of a mental disorder. In some embodiments, the polynucleotide comprises a nucleotide sequence listed in Table 3-6. In some embodiment, the expression of the polynucleotide is enhanced. In some embodiments, the expression of the polynucleotide is decreased. In some embodiments, the methods further comprise administering the compound to an animal and determining the effect on the animal. In some embodiments, the determining step comprises testing the animal's mental function. In some embodiments, the mental disorder is a mood disorder or a psychotic disorder. In some embodiments, the mood disorder is selected from the group consisting of bipolar disorder and major depression. In some embodiments, the psychotic disorder is schizophrenia.
  • The invention also provides methods of treating a mental disorder in a subject. In some embodiments, the methods comprise the step of administering to the subject a therapeutically effective amount of a compound identified using the methods described above. In some embodiments, the mental disorder is a mood disorder or a psychotic disorder. In some embodiments, the mood disorder is selected from the group consisting of bipolar disorder and major depression. In some embodiments, the psychotic disorder is schizophrenia. In some embodiments, the compound is a small organic molecule, an antibody, an antisense molecule, or a peptide.
  • The invention also provides methods of treating mental illness in a subject, comprising the step of administering to the subject a therapeutically effective amount of a polypeptide, which is encoded by a polypeptide that hybridizes under stringent conditions to a nucleic acid of Table 3-6. In some embodiments, the polypeptide comprises an amino acid sequence encoded by a gene listed in Table 3-6. In some embodiments, the mental illness is a mood disorder or a psychotic disorder. In some embodiments, the psychotic disorder is schizophrenia. In some embodiments, the mood disorder is a bipolar disorder or major depression.
  • The invention also provides methods of treating mental illness in a subject, comprising the step of administering to the subject a therapeutically effective amount of a polypeptide, wherein the polypeptide hybridizes under stringent conditions to a nucleic acid of Table 3-6. In some embodiments, the mental illness is a mood disorder or a psychotic disorder. In some embodiments, the psychotic disorder is schizophrenia. In some embodiments, the mood disorder is a bipolar disorder or major depression.
  • BRIEF DESCRIPTION OF THE DRAWINGS
  • Figure 1 : Rodent FGFR2 ISH. Graph shows mean grayscale (n=6 per group) intensity and standard error bars for 35 S signal for regions indicated for both fluoxetine treated and saline treated rats. The increase in signal in the fluoxetine treated group is significant as determined boy a tow-way ANOVA (treatment, brain region) with p=0.0049, Fisher's PLSD.
  • Figure 2: Figure 2 summarizes differential expression of FGF system transcripts in MDD cortex.
  • Figure 3: Figure 3 shows FGF dysregulation is attenuated by anti-depressant therapy.
  • Figure 4: Figure 4 shows that chronic fluoxetine treatment increases FGFR2 expression in rat forebrain.
  • Figure 5: Figure 5 shows environmental complexity: FGF2 and anxiety-like behavior.
  • Figure 6: Figure 6 shows FGF expression negatively correlates with anxiety-like behavior.
  • Figure 7: Figure 7 shows summarizes FGF and negative affect.
  • Figure 8: Figure 8 shows qRT-PCR validation of microarray data.
  • Figure 9: Figure 9 shows G-protein coupled receptor (GPCR) and ligands dysregulated in anterior cingulated cortex of BP subjects.
  • Figure 10 : shows gene category over-representation analysis of the three downstream GPCR signaling pathways.
  • Figure 11: Figure 11 shows phosphatidylinositol metabolism in BP disorder.
  • Figure 12: Figure 12 shows mitogen activated protein kinase signaling in BP disorder.
  • Figure 13: Figure 13 shows cAMP signaling pathway in BP subjects
  • Figure 14: Figure 14 shows genomic structure of the PSPHL gene and the deletion polymorphism of PSPHL that is related to BP susceptibility.
  • Figure 15: Figure 15 shows the predicted amino acid sequences for PSPH, PSPHL-A, and PSPHL-B.
  • Figure 16: Figure 16 shows the serine amino acid metabolic pathway.
  • Figure 17: Figure 17 shows where each exon begins and ends in the PSPHL mRNA and provides primers to detect insertion/deletion polymorphisms in the PSPHL locus.
  • Figure 18: Figure 18 shows a gel image for PSPHL insertion/deletion alleles.
  • Figure 19: Figure 19 shows the cAMP signaling pathway in the limbic system for BP.
  • Figure 20: Figure 20 shows the PI signaling pathwayi in the limbic system for MDD.
  • Figure 21: Figure 21 shows MAPK signaling pathway in the limbic system for MDD.
  • Figure 22: Figure 22 shows the effects of environmental complexity on differences in anxiety behavior and FGF2 gene expression.
  • Figure 23: Figure 23 shows the effects of postnatal FGF2 administration on neurogenesis, emotionality and gene expression in adult rats.
  • Figure 24: Figure 24 shows GABA/glutamate signaling pathways in BP and MDD.
  • Figure 25: Figure 25 shows NCAM SNPs and splice variants involved in mood disorders such as bipolar disorder.
  • Figure 26 : The figure summarizes differential expressed genes regulating cAMP- (A, B) and phosphatidylinositol- (C, D) signaling pathways in the brain of BPD (A, C) and MDD (B, D). GNAI1: G protein alpha inhibiting activity 1, RGS20: Regulator of G-protein signaling 20, PDE1A: Phosphodiesterase 1A, PDE8A: Phosphodiesterase 8A, PKIA: Protein kinase A inhibitor alpha, CDK5: Cyclin-dependent kinase 5, PPP1CA: Protein phosphatase 1, catalytic alpha, PPP1R3C: Protein phosphatase 1, regulatory 3C, INPP5A: Inositol polyphosphate-5-phosphatase A, INPP5F: Inositol polyphosphate-5-phosphatase F, ITPKB: Inositol 1,4,5-trisphosphate 3-kinase B, INPP1: Inositol polyphosphate-1-phosphatase, CDS1: CDP-diacylglycerol synthase 1, PIK3C2A: Phosphoinositide-3-kinase catalytic 2A, PIK3C2B: Phosphoinositide-3-kinase catalytic 2B, PIK3R1: Phosphoinositide-3-kinase regulatory 1, PRKCI: Protein kinase C iota, TTPR1: Inositol 1,4,5-triphosphate receptor 1, PRKB1: Protein kinase C beta 1, NPY: Neuropeptide Y, SST: Somatostatin, NPY1R: Neuropeptide Y receptor Y1, TACR2: Tachykinin receptor 2, NTSR2: Neurotensin receptor 2, EDNRB: Endothelin receptor type B, GRM3: Metabotropic Glutamate receptor 3, EDG1: Endothelial differentiation GPCR 1, EDG2: Endothelial differentiation GPCR 2.
  • Figure 27 : In situ hybridization images of GPR37 mRNA in representative BPD, MDD and control subjects GPR37 mRNA is preferentially expressed in subcortical white matter. Among 6 layers of cortical gray matter, GPR37 expression in deeper layer (V-VI) is relatively higher than superficial layer (I-III). Expression levels in GPR37 is increased in the BPD subjects, and decreased in MDD subjects in subcortical white matter in anterior cingulate cortex tissue, compared to control subjects. WM: Subcortical white matter, BPD: Bipolar disorder, MDD: Major depressive disorder.
  • Figures 28 and 29 : In situ hybridization for LRPPRC (leucine-rich PPR-motif containing) mRNA in three brain regions. (A). LRPPRC expression in BPD and control representative images. (B). Controls with agonal factors showed a 36% reduction in LRPPRC compared to controls with zero agonal factors (p = 0.011) in cerebellum and a similar effect was seen across the cortical regions. (C). In DLPFC, the BPD cases without agonal factors show increased LRPPRC compared to controls without agonal factors (p = 0.001) and compared to MDD subjects (p = 0.02) without agonal factors.
  • Figure 30 : NCAM1 (i.e., neural cell adhesion molecule 1) genomic organization and location of four polymorphic sites. The gene spans 214 kb, but does not contain any exonic SNPs. The arrows indicate the location of the four polymorphisms and the five exons used in this exploratory analysis.
  • Figure 31 : Significant alterations of NCAM1 exon splice variant levels are shown by genotype and diagnosis by genotype.
  • TABLE LEGENDS
  • Table 1a: Table 1a lists subject data for cohort A.
  • Table 1b: Table 1b lists subject data for cohort B.
  • Table 2: Table 2 shows microarray data for all FGF transcripts detected in either DLPFC or AnCg and summary data for confirmation studies.
  • Table 3: Table 3 lists genes uniquely expressed in BP subjects.
  • Table 4: Table 4 lists genes uniquely expressed in MDD subjects.
  • Table 5: Table 5 lists growth factor pathway genes expressed in MDD and BP subjects.
  • Table 6: Table 6 lists GPCR pathway genes expressed in MDD and BP subjects.
  • Table 7: Table 7 lists growth factor pathway genes expressed in MDD and BP subjects.
  • Table 8: Table 8 lists GABA and glutamate signaling pathway genes expressed in MDD and BP subjects.
  • Table 9: Table 9 lists GPCR pathway genes expressed in MDD and BP subjects.
  • Table 10: Table 10 lists mitochondrial genes expressed in MDD and BP subjects.
  • Table 11: Table 11 lists genes expressed in MDD, BP, and schizophrenia subjects.
  • Table 12: Table 12 lists genes expressed in MDD, BP, and schizophrenia subjects.
  • Table 13: Table 13 lists GPCR pathway genes expressed in MDD and BP.
  • Table 14: GPCRs and related signaling genes dysregulated in anterior cingulate cortex.
  • Table 15: GPCRs and related signaling genes dysregulated in dorsolateral prefrontal cortex.
  • Table 16: GPCRs and related signaling genes dysregulated in cerebellar cortex.
  • Table 17: Quantitative RT-PCR data. Fold changes in microarray and qRT-PCR analyses for representative ligand peptides, GPCRs, G protein regulator (NPY, SST, GPR37, GPRC5B, RGS20), which were dysregulated in BPD/MDD compared to the control group. N.S., No significant change; *. p<0.05; **, p<0.01.
  • Table 18: GPCRs and related signaling genes dysregulated in amygdala, hippocampus, nucleus accumbens of BPD.
  • Table 19: GPCRs and related signaling genes dysregulated in amygdala, hippocampus, nucleus accumbens of MDD.
  • Table 20: Table 20 shows the genes that were differentially expressed in BPD or MDD by > 1.2 fold change and were down-regulated in agonal factor control comparisons by < 1.0. The opposite genes are also shown, where there was a decrease in mood disorder by <-1.2 fold change, and the agonal factor control comparison showed an increase > 1.0 fold change. These genes were found in 4 major classifications listed: mitochondria, chaperone, apoptosis, and proteasome.
  • Table 21: Real time Q-PCR validation results for selected mitochondrial related candidate genes for mood disorders in two cortical regions. These genes are nuclear-encoded. Significant by Q-PCR p < 0.05 one-tailed t-test. The Q-PCR t-test MDD, BPD, and control groups used subjects with no agonal factors and pH > 6.8 similar to microarray analysis #3 groups MDD-High, BPD-High, and Control-High.
  • Table 22: Mitochondrial DNA (mtDNA) encoded genes were analyzed by real time Q-PCR for differential expression in BPD and MDD compared to controls. Nuclear encoded genes in BPD and MDD subjects appeared to generally be increased while several mtDNA genes showed a significant decrease by Q-PCR in mood disorders.
  • Table 23: Primers for each DNA segment and possible combination of splice variants (a, b, c, SEC and VASE), as well as, for SNP 9 and an exon outside of the splice sites for measuring total NCAM1. The numbering is shown in Figure 26 according to accession M22094. *1 - Only the forward primer could be designed because exon a is 14 bps. 2 - Exon 3 is before the variable exons and only the forward primer was needed to PCR outside the exons. 3 - Exon 8 is after the variable exons and only the reverse primer was needed to PCR outside the exons.
  • Table 24: Genotypic Association Results. The odds ratio, chi-square (chi2) and p-values where all calculated using the DeFinetti program Tests for Deviation from HWE and Tests for Association (C.I.: 95% confidence interval).
  • Table 25: SNP 9 and SNP b haplotype frequency, odds ratio and p-values. P-values were calculated from the Chi-squared values derived from the EHplus program.
  • Table 26: Genotypic and Allelic Distributions for Controls, Bipolar Disorder and Schizophrenia. Fisher's exact p-values are shown for allelic distribution between case-controls.
  • Table 27: Genotype x Splice Variant Differences x Diagnosis (p-values). For each SNP genotype and splice variant the splice variant amounts were evaluated by t-test based on diagnosis and the significant p-values were reported.
  • Tables 28: Genes upregulated (28.1) and downregulated (28.2) by Lithium in monkey brains.
  • Table 29: Values of V-ATPase Subunits differential expression in Non-human primate model of depression.
  • Table 30: Genes differentially expressed in the frontal cortex of rats subjected to chronic unpredictable stress (CUS) and antidepressant administration (All = fluoxetine, desipramine, and bupropion). Controls were administered water (H2O treated).
  • DEFINITIONS
  • A "mental disorder" or "mental illness" or "mental disease" or "psychiatric or neuropsychiatric disease or illness or disorder" refers to mood disorders (e.g., major depression, mania, and bipolar disorders), psychotic disorders (e.g., schizophrenia, schizoaffective disorder, schizophreniform disorder, delusional disorder, brief psychotic disorder, and shared psychotic disorder), personality disorders, anxiety disorders (e.g., obsessive-compulsive disorder) as well as other mental disorders such as substance -related disorders, childhood disorders, dementia, autistic disorder, adjustment disorder, delirium, multi-infarct dementia, and Tourette's disorder as described in Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, (DSM IV). Typically, such disorders have a complex genetic and/or a biochemical component.
  • A "mood disorder" refers to disruption of feeling tone or emotional state experienced by an individual for an extensive period of time. Mood disorders include major depression disorder (i.e., unipolar disorder), mania, dysphoria, bipolar disorder, dysthymia, cyclothymia and many others. See, e.g., Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, (DSM IV).
  • "Major depression disorder," "major depressive disorder," or "unipolar disorder" refers to a mood disorder involving any of the following symptoms: persistent sad, anxious, or "empty" mood; feelings of hopelessness or pessimism; feelings of guilt, worthlessness, or helplessness; loss of interest or pleasure in hobbies and activities that were once enjoyed, including sex; decreased energy, fatigue, being "slowed down"; difficulty concentrating, remembering, or making decisions; insomnia, early-morning awakening, or oversleeping; appetite and/or weight loss or overeating and weight gain; thoughts of death or suicide or suicide attempts; restlessness or irritability; or persistent physical symptoms that do not respond to treatment, such as headaches, digestive disorders, and chronic pain. Various subtypes of depression are described in, e.g., DSM IV.
  • "Bipolar disorder" is a mood disorder characterized by alternating periods of extreme moods. A person with bipolar disorder experiences cycling of moods that usually swing from being overly elated or irritable (mania) to sad and hopeless (depression) and then back again, with periods of normal mood in between. Diagnosis of bipolar disorder is described in, e.g., DSM IV. Bipolar disorders include bipolar disorder I (mania with or without major depression) and bipolar disorder II (hypomania with major depression), see, e.g., DSM IV.
  • "A psychotic disorder" refers to a condition that affects the mind, resulting in at least some loss of contact with reality. Symptoms of a psychotic disorder include, e.g., hallucinations, changed behavior that is not based on reality, delusions and the like. See, e.g., DSM IV. Schizophrenia, schizoaffective disorder, schizophreniform disorder, delusional disorder, brief psychotic disorder, substance-induced psychotic disorder, and shared psychotic disorder are examples of psychotic disorders.
  • "Schizophrenia" refers to a psychotic disorder involving a withdrawal from reality by an individual. Symptoms comprise for at least a part of a month two or more of the following symptoms: delusions (only one symptom is required if a delusion is bizarre, such as being abducted in a space ship from the sun); hallucinations (only one symptom is required if hallucinations are of at least two voices talking to one another or of a voice that keeps up a running commentary on the patient's thoughts or actions); disorganized speech (e.g., frequent derailment or incoherence); grossly disorganized or catatonic behavior; or negative symptoms, i.e., affective flattening, alogia, or avolition. Schizophrenia encompasses disorders such as, e.g., schizoaffective disorders. Diagnosis of schizophrenia is described in, e.g., DSM IV. Types of schizophrenia include, e.g., paranoid, disorganized, catatonic, undifferentiated, and residual.
  • An "antidepressant" refers to an agents typically used to treat clinical depression. Antidepressants includes compounds of different classes including, for example, specific serotonin reuptake inhibitors (e.g., fluoxetine), tricyclic antidepressants (e.g., desipramine), and dopamine reuptake inhibitors (e.g, bupropion). Typically, antidepressants of different classes exert their therapeutic effects via different biochemical pathways. Often these biochemical pathways overlap or intersect. Additonal diseases or disorders often treated with antidepressants include, chronic pain, anxiety disorders, and hot flashes.
  • An "agonist" refers to an agent that binds to a polypeptide or polynucleotide of the invention, stimulates, increases, activates, facilitates, enhances activation, sensitizes or up regulates the activity or expression of a polypeptide or polynucleotide of the invention.
  • An "antagonist" refers to an agent that inhibits expression of a polypeptide or polynucleotide of the invention or binds to, partially or totally blocks stimulation, decreases, prevents, delays activation, inactivates, desensitizes, or down regulates the activity of a polypeptide or polynucleotide of the invention.
  • "Inhibitors," "activators," and "modulators" of expression or of activity are used to refer to inhibitory, activating, or modulating molecules, respectively, identified using in vitro and in vivo assays for expression or activity, e.g., ligands, agonists, antagonists, and their homologs and mimetics. The term "modulator" includes inhibitors and activators. Inhibitors are agents that, e.g., inhibit expression of a polypeptide or polynucleotide of the invention or bind to, partially or totally block stimulation or enzymatic activity, decrease, prevent, delay activation, inactivate, desensitize, or down regulate the activity of a polypeptide or polynucleotide of the invention, e.g., antagonists. Activators are agents that, e.g., induce or activate the expression of a polypeptide or polynucleotide of the invention or bind to, stimulate, increase, open, activate, facilitate, enhance activation or enzymatic activity, sensitize or up regulate the activity of a polypeptide or polynucleotide of the invention, e.g., agonists. Modulators include naturally occurring and synthetic ligands, antagonists, agonists, small chemical molecules and the like. Assays to identify inhibitors and activators include, e.g., applying putative modulator compounds to cells, in the presence or absence of a polypeptide or polynucleotide of the invention and then determining the functional effects on a polypeptide or polynucleotide of the invention activity. Samples or assays comprising a polypeptide or polynucleotide of the invention that are treated with a potential activator, inhibitor, or modulator are compared to control samples without the inhibitor, activator, or modulator to examine the extent of effect. Control samples (untreated with modulators) are assigned a relative activity value of 100%. Inhibition is achieved when the activity value of a polypeptide or polynucleotide of the invention relative to the control is about 80%, optionally 50% or 25-1%. Activation is achieved when the activity value of a polypeptide or polynucleotide of the invention relative to the control is 110%, optionally 150%, optionally 200-500%, or 1000-3000% higher.
  • The term "test compound" or "drug candidate" or "modulator" or grammatical equivalents as used herein describes any molecule, either naturally occurring or synthetic, e.g., protein, oligopeptide (e.g., from about 5 to about 25 amino acids in length, preferably from about 10 to 20 or 12 to 18 amino acids in length, preferably 12, 15, or 18 amino acids in length), small organic molecule, polysaccharide, lipid, fatty acid, polynucleotide, RNAi, oligonucleotide, etc. The test compound can be in the form of a library of test compounds, such as a combinatorial or randomized library that provides a sufficient range of diversity. Test compounds are optionally linked to a fusion partner, e.g., targeting compounds, rescue compounds, dimerization compounds, stabilizing compounds, addressable compounds, and other functional moieties. Conventionally, new chemical entities with useful properties are generated by identifying a test compound (called a "lead compound") with some desirable property or activity, e.g., inhibiting activity, creating variants of the lead compound, and evaluating the property and activity of those variant compounds. Often, high throughput screening (HTS) methods are employed for such an analysis.
  • A "small organic molecule" refers to an organic molecule, either naturally occurring or synthetic, that has a molecular weight of more than about 50 Daltons and less than about 2500 Daltons, preferably less than about 2000 Daltons, preferably between about 100 to about 1000 Daltons, more preferably between about 200 to about 500 Daltons.
  • An "siRNA" or "RNAi" refers to a nucleic acid that forms a double stranded RNA, which double stranded RNA has the ability to reduce or inhibit expression of a gene or target gene when the siRNA expressed in the same cell as the gene or target gene. "siRNA" or "RNAi" thus refers to the double stranded RNA formed by the complementary strands. The complementary portions of the siRNA that hybridize to form the double stranded molecule typically have substantial or complete identity. In one embodiment, an siRNA refers to a nucleic acid that has substantial or complete identity to a target gene and forms a double stranded siRNA. Typically, the siRNA is at least about 15-50 nucleotides in length (e.g., each complementary sequence of the double stranded siRNA is 15-50 nucleotides in length, and the double stranded siRNA is about 15-50 base pairs in length, preferable about preferably about 20-30 base nucleotides, preferably about 20-25 or about 24-29 nucleotides in length, e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.
  • "Determining the functional effect" refers to assaying for a compound that increases or decreases a parameter that is indirectly or directly under the influence of a polynucleotide or polypeptide of the invention (such as a polynucleotide of Table 3-6 or a polypeptide encoded by a gene of Table 3-6), e.g., measuring physical and chemical or phenotypic effects. Such functional effects can be measured by any means known to those skilled in the art, e.g., changes in spectroscopic (e.g., fluorescence, absorbance, refractive index), hydrodynamic (e.g., shape), chromatographic, or solubility properties for the protein; measuring inducible markers or transcriptional activation of the protein; measuring binding activity or binding assays, e.g. binding to antibodies; measuring changes in ligand binding affinity; measurement of calcium influx; measurement of the accumulation of an enzymatic product of a polypeptide of the invention or depletion of an substrate; measurement of changes in protein levels of a polypeptide of the invention; measurement of RNA stability; G-protein binding; GPCR phosphorylation or dephosphorylation; signal transduction, e.g., receptor-ligand interactions, second messenger concentrations (e.g., cAMP, IP3, or intracellular Ca2+); identification of downstream or reporter gene expression (CAT, luciferase, β-gal, GFP and the like), e.g., via chemiluminescence, fluorescence, colorimetric reactions, antibody binding, inducible markers, and ligand binding assays.
  • Samples or assays comprising a nucleic acid or protein disclosed herein that are treated with a potential activator, inhibitor, or modulator are compared to control samples without the inhibitor, activator, or modulator to examine the extent of inhibition. Control samples (untreated with inhibitors) are assigned a relative protein activity value of 100%. Inhibition is achieved when the activity value relative to the control is about 80%, preferably 50%, more preferably 25-0%. Activation is achieved when the activity value relative to the control (untreated with activators) is 110%, more preferably 150%, more preferably 200-500% (i.e., two to five fold higher relative to the control), more preferably 1000-3000% higher.
  • "Biological sample" includes sections of tissues such as biopsy and autopsy samples, and frozen sections taken for histologic purposes. Such samples include blood, sputum, tissue, lysed cells, brain biopsy, cultured cells, e.g., primary cultures, explants, and transformed cells, stool, urine, etc. A biological sample is typically obtained from a eukaryotic organism, most preferably a mammal such as a primate, e.g., chimpanzee or human; cow; dog; cat; a rodent, e.g., guinea pig, rat, mouse; rabbit; or a bird; reptile; or fish.
  • "Antibody" refers to a polypeptide substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof which specifically bind and recognize an analyte (antigen). The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
  • An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains respectively.
  • Antibodies exist, e.g., as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)'2, a dimer of Fab which itself is a light chain joined to VH-C H1 by a disulfide bond. The F(ab)'2 may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)'2 dimer into an Fab' monomer. The Fab' monomer is essentially an Fab with part of the hinge region (see, Paul (Ed.) Fundamental Immunology, Third Edition, Raven Press, NY (1993)). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by utilizing recombinant DNA methodology. Thus, the term antibody, as used herein, also includes antibody fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv).
  • The terms "peptidomimetic" and "mimetic" refer to a synthetic chemical compound that has substantially the same structural and functional characteristics of the polynucleotides, polypeptides, antagonists or agonists of the invention. Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties analogous to those of the template peptide. These types of non-peptide compound are termed "peptide mimetics" or "peptidomimetics" (Fauchere, Adv. Drug Res. 15:29 (1986); Veber and Freidinger TINS p. 392 (1985); and Evans et al., J. Med. Chem. 30:1229 (1987), which are incorporated herein by reference). Peptide mimetics that are structurally similar to therapeutically useful peptides may be used to produce an equivalent or enhanced therapeutic or prophylactic effect. Generally, peptidomimetics are structurally similar to a paradigm polypeptide (i.e., a polypeptide that has a biological or pharmacological activity), such as a CCX CKR, but have one or more peptide linkages optionally replaced by a linkage selected from the group consisting of, e.g., -CH2NH-, -CH2S-, -CH2-CH2-, -CH=CH- (cis and trans), - COCH2-, -CH(OH)CH2-, and -CH2SO-. The mimetic can be either entirely composed of synthetic, non-natural analogues of amino acids, or, is a chimeric molecule of partly natural peptide amino acids and partly non-natural analogs of amino acids. The mimetic can also incorporate any amount of natural amino acid conservative substitutions as long as such substitutions also do not substantially alter the mimetic's structure and/or activity. For example, a mimetic composition is within the scope of the invention if it is capable of carrying out the binding or enzymatic activities of a polypeptide or polynucleotide of the invention or inhibiting or increasing the enzymatic activity or expression of a polypeptide or polynucleotide of the invention.
  • The term "gene" means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons).
  • The term "isolated," when applied to a nucleic acid or protein, denotes that the nucleic acid or protein is essentially free of other cellular components with which it is associated in the natural state. It is preferably in a homogeneous state although it can be in either a dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein that is the predominant species present in a preparation is substantially purified. In particular, an isolated gene is separated from open reading frames that flank the gene and encode a protein other than the gene of interest. The term "purified" denotes that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. Particularly, it means that the nucleic acid or protein is at least 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure.
  • The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Cassol et al. (1992); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The term nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene.
  • The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. As used herein, the terms encompass amino acid chains of any length, including full-length proteins (i.e., antigens), wherein the amino acid residues are linked by covalent peptide bonds.
  • The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. "Amino acid mimetics" refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
  • Amino acids may be referred to herein by either the commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
  • "Conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, "conservatively modified variants" refers to those nucleic acids that encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein that encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence.
  • As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.
  • The following eight groups each contain amino acids that are conservative substitutions for one another:
    1. 1) Alanine (A), Glycine (G);
    2. 2) Aspartic acid (D), Glutamic acid (E);
    3. 3) Asparagine (N), Glutamine (Q);
    4. 4) Arginine (R), Lysine (K);
    5. 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);
    6. 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);
    7. 7) Serine (S), Threonine (T); and
    8. 8) Cysteine (C), Methionine (M)
      (see, e.g., Creighton, Proteins (1984)).
  • "Percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
  • The terms "identical" or percent "identity," in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity over a specified region), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Such sequences are then said to be "substantially identical." This definition also refers to the complement of a test sequence. Optionally, the identity exists over a region that is at least about 50 nucleotides in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides in length.
  • For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
  • A "comparison window", as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman (1988) Proc. Nat'l. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, e.g., Ausubel et al., Current Protocols in Molecular Biology (1995 supplement)).
  • An example of an algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nuc. Acids Res. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) or 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.
  • The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.
  • An indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.
  • The phrase "selectively (or specifically) hybridizes to" refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent hybridization conditions when that sequence is present in a complex mixture (e.g., total cellular or library DNA or RNA).
  • The phrase "stringent hybridization conditions" refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acid, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays" (1993). Generally, stringent conditions are selected to be about 5-10° C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH. The Tm is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium). Stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least two times background, optionally 10 times background hybridization. Exemplary stringent hybridization conditions can be as following: 50% formamide, 5X SSC, and 1% SDS, incubating at 42°C, or 5X SSC, 1% SDS, incubating at 65°C, with wash in 0.2X SSC, and 0.1% SDS at 65°C. Such washes can be performed for 5, 15, 30, 60, 120, or more minutes. Nucleic acids that hybridize to the genes listed in Tables 3-10 and Figure 14 are encompassed by the invention.
  • Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides that they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cases, the nucleic acids typically hybridize under moderately stringent hybridization conditions. Exemplary "moderately stringent hybridization conditions" include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37°C, and a wash in 1X SSC at 45°C. Such washes can be performed for 5, 15, 30, 60, 120, or more minutes. A positive hybridization is at least twice background. Those of ordinary skill will readily recognize that alternative hybridization and wash conditions can be utilized to provide conditions of similar stringency.
  • For PCR, a temperature of about 36°C is typical for low stringency amplification, although annealing temperatures may vary between about 32°C and 48°C depending on primer length. For high stringency PCR amplification, a temperature of about 62°C is typical, although high stringency annealing temperatures can range from about 50°C to about 65°C, depending on the primer length and specificity. Typical cycle conditions for both high and low stringency amplifications include a denaturation phase of 90°C - 95°C for 30 sec - 2 min., an annealing phase lasting 30 sec. - 2 min., and an extension phase of about 72°C for 1 - 2 min. Protocols and guidelines for low and high stringency amplification reactions are provided, e.g., in Innis et al., PCR Protocols, A Guide to Methods and Applications (1990).
  • The phrase "a nucleic acid sequence encoding" refers to a nucleic acid that contains sequence information for a structural RNA such as rRNA, a tRNA, or the primary amino acid sequence of a specific protein or peptide, or a binding site for a trans-acting regulatory agent. This phrase specifically encompasses degenerate codons (i.e., different codons which encode a single amino acid) of the native sequence or sequences which may be introduced to conform with codon preference in a specific host cell.
  • The term "recombinant" when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (nonrecombinant) form of the cell or express native genes that are otherwise abnormally expressed, under-expressed or not expressed at all.
  • The term "heterologous" when used with reference to portions of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature. For instance, the nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).
  • An "expression vector" is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a host cell. The expression vector can be part of a plasmid, virus, or nucleic acid fragment. Typically, the expression vector includes a nucleic acid to be transcribed operably linked to a promoter.
  • The phrase "specifically (or selectively) binds to an antibody" or "specifically (or selectively) immunoreactive with", when referring to a protein or peptide, refers to a binding reaction which is determinative of the presence of the protein in the presence of a heterogeneous population of proteins and other biologics. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein and do not bind in a significant amount to other proteins present in the sample. Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein. For example, antibodies raised against a protein having an amino acid sequence encoded by any of the polynucleotides of the invention can be selected to obtain antibodies specifically immunoreactive with that protein and not with other proteins, except for polymorphic variants. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays, Western blots, or immunohistochemistry are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See, Harlow and Lane Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, NY (1988) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity. Typically, a specific or selective reaction will be at least twice the background signal or noise and more typically more than 10 to 100 times background.
  • One who is "predisposed for a mental disorder" as used herein means a person who has an inclination or a higher likelihood of developing a mental disorder when compared to an average person in the general population.
  • DETAILED DESCRIPTION OF THE INVENTION I. Introduction
  • To understand the genetic basis of mental disorders, studies have been conducted to investigate the expression patterns of genes that are differentially expressed specifically in central nervous system of subjects with mood disorders. Differential and unique expression of known and novel genes was determined by way of interrogating total RNA samples purified from postmortem brains of BP and MDD patients with Affymetrix Gene Chips® (containing high-density oligonucleotide probe set arrays). The fundamental principles is that by identifying genes and pathways that are differentially expressed in BP and/or MDD (relative to healthy control subjects), via global expression profiling of the transcriptomes as above, one can identify genes that cause, effect, or are associated with the disease, or that interact with drugs used to treat the disease, for use in diagnostic and therapeutic applications.
  • The present invention therefore demonstrates the altered expression (either higher or lower expression as indicated herein) and in some cases unique differential expression of the genes of Tables 3-10 at the mRNA level in selected brain regions of patients diagnosed with mood disorders, as well as the PSPHL gene (see, e.g., Figure 14) (e.g., bipolar disorder and major depression disorder) in comparison with normal individuals. This invention thus provides methods for diagnosis of mental disorders such as mood disorders (e.g., bipolar disorder, major depression, and the like) and other mental disorders having a genetic component by detecting the level of a transcript or translation product of the genes listed in Tables 3-10 and Figure 14 as well as their corresponding biochemical pathways.
  • In one embodiment, the present invention relates to a novel insertion-deletion polymorphism of phosphoserine phosphatase-like gene, and the association between deletion allele of PSPHL and susceptibility to bipolar disorder (BPD). The fact that PSPHL shows dichotomous present/absent pattern of expression among individuals with brain-wide consistency suggests genetic variation in its regulation (see Example 2). Most intriguingly, we have identified an insertion/deletion polymorphism at the PSPHL locus. The deleted genomic region spans more than 30 kb, including the promoter region and the exons 1, 2 and 3 of PSPHL gene. This genetic variance explains the present/absent pattern of the PSPHL expression. An over-representation of the deletion allele resulting in the absence of PSPHL expression increases susceptibility to BPD. The invention therefore provides the first evidence linking a genetic variant of the PSPHL gene to bipolar disorder. The finding will facilitate characterization of the physiological and pathological function of the gene relevant to bipolar disorder, and provides novel and significant use of this gene and its variants for diagnosis, treatment and prevention of bipolar disorder.
  • The invention further provides methods of identifying a compound useful for the treatment of such disorders by selecting compounds that modulates the functional effect of the translation products or the expression of the transcripts described herein. The invention also provides for methods of treating patients with such mental disorders, e.g., by administering the compounds of the invention or by gene therapy.
  • The genes and the polypeptides that they encode, which are associated with mood disorders such as bipolar disease and major depression, are useful for facilitating the design and development of various molecular diagnostic tools such as GeneChips™ containing probe sets specific for all or selected mental disorders, including but not limited to mood disorders, and as an ante-and/or post-natal diagnostic tool for screening newborns in concert with genetic counseling. Other diagnostic applications include evaluation of disease susceptibility, prognosis, and monitoring of disease or treatment process, as well as providing individualized medicine via predictive drug profiling systems, e.g., by correlating specific genomic motifs with the clinical response of a patient to individual drugs. In addition, the present invention is useful for multiplex SNP and haplotype profiling, including but not limited to the identification of therapeutic, diagnostic, and pharmacogenetic targets at the gene, mRNA, protein, and pathway level. Profiling of splice variants and deletions is also useful for diagnostic and therapeutic applications.
  • The genes and the polypeptides that they encode, described herein, are also useful as drug targets for the development of therapeutic drugs for the treatment or prevention of mental disorders, including but not limited to mood disorders.
  • Antidepressants belong to different classes, e.g., desipramine, bupropion, and fluoxetine are in general equally effective for the treatment of clinical depression, but act by different mechanisms. The similar effectiveness of the drugs for treatment of mood disorders suggests that they act through a presently unidentified common pathway. Animal models of depression, including treatment of animals with known therapeutics such as SSRIs, can be used to examine the mode of action of the genes of the invention. Lithium is drug of choice for treating BP.
  • The genes and the polypeptides that they encode, described herein, as also useful as drug targets for the development of therapeutic drugs for the treatment or prevention of mental disorders, including but not limited to mood disorders. Mental disorders have a high co-morbidity with other neurological disorders, such as Parkinson's disease or Alzheimer's. Therefore, the present invention can be used for diagnosis and treatment of patients with multiple disease states that include a mental disorder such as a mood disorder. These mood disorders include BP, MDD, and other disorders such as psychotic-depression, depression and anxiety features, melancholic depression, chronic depression, BPI and BPII.
  • II. General Recombinant nucleic acid methods for use with the invention
  • In numerous embodiments of the present invention, polynucleotides of the invention will be isolated and cloned using recombinant methods. Such polynucleotides include, e.g., those listed in Tables 3-10 and Figure 14, which can be used for, e.g., protein expression or during the generation of variants, derivatives, expression cassettes, to monitor gene expression, for the isolation or detection of sequences of the invention in different species, for diagnostic purposes in a patient, e.g., to detect mutations or to detect expression levels of nucleic acids or polypeptides of the invention. In some embodiments, the sequences of the invention are operably linked to a heterologous promoter. In one embodiment, the nucleic acids of the invention are from any mammal, including, in particular, e.g., a human, a mouse, a rat, a primate, etc.
  • A. General Recombinant Nucleic Acids Methods
  • This invention relies on routine techniques in the field of recombinant genetics. Basic texts disclosing the general methods of use in this invention include Sambrook et al., Molecular Cloning, A Laboratory Manual (3rd ed. 2001); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al., eds., 1994)).
  • For nucleic acids, sizes are given in either kilobases (kb) or base pairs (bp). These are estimates derived from agarose or acrylamide gel electrophoresis, from sequenced nucleic acids, or from published DNA sequences. For proteins, sizes are given in kilodaltons (kDa) or amino acid residue numbers. Proteins sizes are estimated from gel electrophoresis, from sequenced proteins, from derived amino acid sequences, or from published protein sequences.
  • Oligonucleotides that are not commercially available can be chemically synthesized according to the solid phase phosphoramidite triester method first described by Beaucage & Caruthers, Tetrahedron Letts. 22:1859-1862 (1981), using an automated synthesizer, as described in Van Devanter et. al., Nucleic Acids Res. 12:6159-6168 (1984). Purification of oligonucleotides is by either native acrylamide gel electrophoresis or by anion-exchange HPLC as described in Pearson & Reanier, J. Chrom. 255:137-149 (1983).
  • The sequence of the cloned genes and synthetic oligonucleotides can be verified after cloning using, e.g., the chain termination method for sequencing double-stranded templates of Wallace et al., Gene 16:21-26 (1981).
  • B. Cloning Methods for the Isolation of Nucleotide Sequences Encoding Desired Proteins
  • In general, the nucleic acids encoding the subject proteins are cloned from DNA sequence libraries that are made to encode cDNA or genomic DNA. The particular sequences can be located by hybridizing with an oligonucleotide probe, the sequence of which can be derived from the sequences of the genes listed in Tables 3-10 and Figure 14, which provide a reference for PCR primers and defines suitable regions for isolating specific probes. Alternatively, where the sequence is cloned into an expression library, the expressed recombinant protein can be detected immunologically with antisera or purified antibodies made against a polypeptide comprising an amino acid sequence encoded by a gene listed in Table 1-8.
  • Methods for making and screening genomic and cDNA libraries are well known to those of skill in the art (see, e.g., Gubler and Hoffman Gene 25:263-269 (1983); Benton and Davis Science, 196:180-182 (1977); and Sambrook, supra). Brain cells are an example of suitable cells to isolate RNA and cDNA sequences of the invention.
  • Briefly, to make the cDNA library, one should choose a source that is rich in mRNA. The mRNA can then be made into cDNA, ligated into a recombinant vector, and transfected into a recombinant host for propagation, screening and cloning. For a genomic library, the DNA is extracted from a suitable tissue and either mechanically sheared or enzymatically digested to yield fragments of preferably about 5-100 kb. The fragments are then separated by gradient centrifugation from undesired sizes and are constructed in bacteriophage lambda vectors. These vectors and phage are packaged in vitro, and the recombinant phages are analyzed by plaque hybridization. Colony hybridization is carried out as generally described in Grunstein et al., Proc. Natl. Acad. Sci. USA., 72:3961-3965 (1975).
  • An alternative method combines the use of synthetic oligonucleotide primers with polymerase extension on an mRNA or DNA template. Suitable primers can be designed from specific sequences of the invention. This polymerase chain reaction (PCR) method amplifies the nucleic acids encoding the protein of interest directly from mRNA, cDNA, genomic libraries or cDNA libraries. Restriction endonuclease sites can be incorporated into the primers. Polymerase chain reaction or other in vitro amplification methods may also be useful, for example, to clone nucleic acids encoding specific proteins and express said proteins, to synthesize nucleic acids that will be used as probes for detecting the presence of mRNA encoding a polypeptide of the invention in physiological samples, for nucleic acid sequencing, or for other purposes (see, U.S. Patent Nos. 4,683,195 and 4,683,202 ). Genes amplified by a PCR reaction can be purified from agarose gels and cloned into an appropriate vector.
  • Appropriate primers and probes for identifying polynucleotides of the invention from mammalian tissues can be derived from the sequences provided herein. For a general overview of PCR, see, Innis et al. PCR Protocols: A Guide to Methods and Applications, Academic Press, San Diego (1990).
  • Synthetic oligonucleotides can be used to construct genes. This is done using a series of overlapping oligonucleotides, usually 40-120 bp in length, representing both the sense and anti-sense strands of the gene. These DNA fragments are then annealed, ligated and cloned.
  • A gene encoding a polypeptide of the invention can be cloned using intermediate vectors before transformation into mammalian cells for expression. These intermediate vectors are typically prokaryote vectors or shuttle vectors. The proteins can be expressed in either prokaryotes, using standard methods well known to those of skill in the art, or eukaryotes as described infra.
  • III. Purification of proteins of the invention
  • Either naturally occurring or recombinant polypeptides of the invention can be purified for use in functional assays. Naturally occurring polypeptides, e.g., polypeptides encoded by genes listed in Tables 3-10 and Figure 14, can be purified, for example, from mouse or human tissue such as brain or any other source of an ortholog. Recombinant polypeptides can be purified from any suitable expression system.
  • The polypeptides of the invention may be purified to substantial purity by standard techniques, including selective precipitation with such substances as ammonium sulfate; column chromatography, immunopurification methods, and others (see, e.g., Scopes, Protein Purification: Principles and Practice (1982); U.S. Patent No. 4,673,641 ; Ausubel et al., supra; and Sambrook et al., supra).
  • A number of procedures can be employed when recombinant polypeptides are purified. For example, proteins having established molecular adhesion properties can be reversible fused to polypeptides of the invention. With the appropriate ligand, the polypeptides can be selectively adsorbed to a purification column and then freed from the column in a relatively pure form. The fused protein is then removed by enzymatic activity. Finally the polypeptide can be purified using immunoaffinity columns.
  • A. Purification of Proteins from Recombinant Bacteria
  • When recombinant proteins are expressed by the transformed bacteria in large amounts, typically after promoter induction, although expression can be constitutive, the proteins may form insoluble aggregates. There are several protocols that are suitable for purification of protein inclusion bodies. For example, purification of aggregate proteins (hereinafter referred to as inclusion bodies) typically involves the extraction, separation and/or purification of inclusion bodies by disruption of bacterial cells typically, but not limited to, by incubation in a buffer of about 100-150 µg/ml lysozyme and 0.1% Nonidet P40, a non-ionic detergent. The cell suspension can be ground using a Polytron grinder (Brinkman Instruments, Westbury, NY). Alternatively, the cells can be sonicated on ice. Alternate methods of lysing bacteria are described in Ausubel et al. and Sambrook et al., both supra, and will be apparent to those of skill in the art.
  • The cell suspension is generally centrifuged and the pellet containing the inclusion bodies resuspended in buffer which does not dissolve but washes the inclusion bodies, e.g., 20 mM Tris-HCl (pH 7.2), 1 mM EDTA, 150 mM NaCl and 2% Triton-X 100, a non-ionic detergent. It may be necessary to repeat the wash step to remove as much cellular debris as possible. The remaining pellet of inclusion bodies may be resuspended in an appropriate buffer (e.g., 20 mM sodium phosphate, pH 6.8, 150 mM NaCl). Other appropriate buffers will be apparent to those of skill in the art.
  • Following the washing step, the inclusion bodies are solubilized by the addition of a solvent that is both a strong hydrogen acceptor and a strong hydrogen donor (or a combination of solvents each having one of these properties). The proteins that formed the inclusion bodies may then be renatured by dilution or dialysis with a compatible buffer. Suitable solvents include, but are not limited to, urea (from about 4 M to about 8 M), formamide (at least about 80%, volume/volume basis), and guanidine hydrochloride (from about 4 M to about 8 M). Some solvents that are capable of solubilizing aggregate-forming proteins, such as SDS (sodium dodecyl sulfate) and 70% formic acid, are inappropriate for use in this procedure due to the possibility of irreversible denaturation of the proteins, accompanied by a lack of immunogenicity and/or activity. Although guanidine hydrochloride and similar agents are denaturants, this denaturation is not irreversible and renaturation may occur upon removal (by dialysis, for example) or dilution of the denaturant, allowing re-formation of the immunologically and/or biologically active protein of interest. After solubilization, the protein can be separated from other bacterial proteins by standard separation techniques.
  • Alternatively, it is possible to purify proteins from bacteria periplasm. Where the protein is exported into the periplasm of the bacteria, the periplasmic fraction of the bacteria can be isolated by cold osmotic shock in addition to other methods known to those of skill in the art (see, Ausubel et al., supra). To isolate recombinant proteins from the periplasm, the bacterial cells are centrifuged to form a pellet. The pellet is resuspended in a buffer containing 20% sucrose. To lyse the cells, the bacteria are centrifuged and the pellet is resuspended in ice-cold 5 mM MgSO4 and kept in an ice bath for approximately 10 minutes. The cell suspension is centrifuged and the supernatant decanted and saved. The recombinant proteins present in the supernatant can be separated from the host proteins by standard separation techniques well known to those of skill in the art.
  • B. Standard Protein Separation Techniques For Purifying Proteins 1. Solubility Fractionation
  • Often as an initial step, and if the protein mixture is complex, an initial salt fractionation can separate many of the unwanted host cell proteins (or proteins derived from the cell culture media) from the recombinant protein of interest. The preferred salt is ammonium sulfate. Ammonium sulfate precipitates proteins by effectively reducing the amount of water in the protein mixture. Proteins then precipitate on the basis of their solubility. The more hydrophobic a protein is, the more likely it is to precipitate at lower ammonium sulfate concentrations. A typical protocol is to add saturated ammonium sulfate to a protein solution so that the resultant ammonium sulfate concentration is between 20-30%. This will precipitate the most hydrophobic proteins. The precipitate is discarded (unless the protein of interest is hydrophobic) and ammonium sulfate is added to the supernatant to a concentration known to precipitate the protein of interest. The precipitate is then solubilized in buffer and the excess salt removed if necessary, through either dialysis or diafiltration. Other methods that rely on solubility of proteins, such as cold ethanol precipitation, are well known to those of skill in the art and can be used to fractionate complex protein mixtures.
  • 2. Size Differential Filtration
  • Based on a calculated molecular weight, a protein of greater and lesser size can be isolated using ultrafiltration through membranes of different pore sizes (for example, Amicon or Millipore membranes). As a first step, the protein mixture is ultrafiltered through a membrane with a pore size that has a lower molecular weight cut-off than the molecular weight of the protein of interest. The retentate of the ultrafiltration is then ultrafiltered against a membrane with a molecular cut off greater than the molecular weight of the protein of interest. The recombinant protein will pass through the membrane into the filtrate. The filtrate can then be chromatographed as described below.
  • 3. Column Chromatography
  • The proteins of interest can also be separated from other proteins on the basis of their size, net surface charge, hydrophobicity and affinity for ligands. In addition, antibodies raised against proteins can be conjugated to column matrices and the proteins immunopurified. All of these methods are well known in the art.
  • It will be apparent to one of skill that chromatographic techniques can be performed at any scale and using equipment from many different manufacturers (e.g., Pharmacia Biotech).
  • IV. Detection of gene expression
  • Those of skill in the art will recognize that detection of expression of polynucleotides of the invention has many uses. For example, as discussed herein, detection of the level of polypeptides or polynucleotides of the invention in a patient is useful for diagnosing mood disorders or psychotic disorders or a predisposition for a mood disorder or psychotic disorders. Moreover, detection of gene expression is useful to identify modulators of expression of the polypeptides or polynucleotides of the invention.
  • A variety of methods of specific DNA and RNA measurement using nucleic acid hybridization techniques are known to those of skill in the art (see, Sambrook, supra). Some methods involve an electrophoretic separation (e.g., Southern blot for detecting DNA, and Northern blot for detecting RNA), but measurement of DNA and RNA can also be carried out in the absence of electrophoretic separation (e.g., by dot blot). Southern blot of genomic DNA (e.g., from a human) can be used for screening for restriction fragment length polymorphism (RFLP) to detect the presence of a genetic disorder affecting a polypeptide of the invention.
  • The selection of a nucleic acid hybridization format is not critical. A variety of nucleic acid hybridization formats are known to those skilled in the art. For example, common formats include sandwich assays and competition or displacement assays. Hybridization techniques are generally described in Hames and Higgins Nucleic Acid Hybridization, A Practical Approach, IRL Press (1985); Gall and Pardue, Proc. Natl. Acad. Sci. U.S.A., 63:378-383 (1969); and John et al. Nature, 223:582-587 (1969).
  • Detection of a hybridization complex may require the binding of a signal-generating complex to a duplex of target and probe polynucleotides or nucleic acids. Typically, such binding occurs through ligand and anti-ligand interactions as between a ligand-conjugated probe and an anti-ligand conjugated with a signal. The binding of the signal generation complex is also readily amenable to accelerations by exposure to ultrasonic energy.
  • The label may also allow indirect detection of the hybridization complex. For example, where the label is a hapten or antigen, the sample can be detected by using antibodies. In these systems, a signal is generated by attaching fluorescent or enzyme molecules to the antibodies or in some cases, by attachment to a radioactive label (see, e.g., Tijssen, "Practice and Theory of Enzyme Immunoassays," Laboratory Techniques in Biochemistry and Molecular Biology, Burdon and van Knippenberg Eds., Elsevier (1985), pp. 9-20).
  • The probes are typically labeled either directly, as with isotopes, chromophores, lumiphores, chromogens, or indirectly, such as with biotin, to which a streptavidin complex may later bind. Thus, the detectable labels used in the assays of the present invention can be primary labels (where the label comprises an element that is detected directly or that produces a directly detectable element) or secondary labels (where the detected label binds to a primary label, e.g., as is common in immunological labeling). Typically, labeled signal nucleic acids are used to detect hybridization. Complementary nucleic acids or signal nucleic acids may be labeled by any one of several methods typically used to detect the presence of hybridized polynucleotides. The most common method of detection is the use of autoradiography with 3H, 125I, 35S, 14C, or 32P-labeled probes or the like.
  • Other labels include, e.g., ligands that bind to labeled antibodies, fluorophores, chemiluminescent agents, enzymes, and antibodies which can serve as specific binding pair members for a labeled ligand. An introduction to labels, labeling procedures and detection of labels is found in Polak and Van Noorden Introduction to Immunocytochemistry, 2nd ed., Springer Verlag, NY (1997); and in Haugland Handbook of Fluorescent Probes and Research Chemicals, a combined handbook and catalogue Published by Molecular Probes, Inc. (1996).
  • In general, a detector which monitors a particular probe or probe combination is used to detect the detection reagent label. Typical detectors include spectrophotometers, phototubes and photodiodes, microscopes, scintillation counters, cameras, film and the like, as well as combinations thereof. Examples of suitable detectors are widely available from a variety of commercial sources known to persons of skill in the art. Commonly, an optical image of a substrate comprising bound labeling moieties is digitized for subsequent computer analysis.
  • Most typically, the amount of RNA is measured by quantifying the amount of label fixed to the solid support by binding of the detection reagent. Typically, the presence of a modulator during incubation will increase or decrease the amount of label fixed to the solid support relative to a control incubation which does not comprise the modulator, or as compared to a baseline established for a particular reaction type. Means of detecting and quantifying labels are well known to those of skill in the art.
  • In preferred embodiments, the target nucleic acid or the probe is immobilized on a solid support. Solid supports suitable for use in the assays of the invention are known to those of skill in the art. As used herein, a solid support is a matrix of material in a substantially fixed arrangement.
  • A variety of automated solid-phase assay techniques are also appropriate. For instance, very large scale immobilized polymer arrays (VLSIPS™), available from Affymetrix, Inc. (Santa Clara, CA) can be used to detect changes in expression levels of a plurality of genes involved in the same regulatory pathways simultaneously. See, Tijssen, supra., Fodor et al. (1991) Science, 251: 767- 777; Sheldon et al. (1993) Clinical Chemistry 39(4): 718-719, and Kozal et al. (1996) Nature Medicine 2(7): 753-759.
  • Detection can be accomplished, for example, by using a labeled detection moiety that binds specifically to duplex nucleic acids (e.g., an antibody that is specific for RNA-DNA duplexes). One preferred example uses an antibody that recognizes DNA-RNA heteroduplexes in which the antibody is linked to an enzyme (typically by recombinant or covalent chemical bonding). The antibody is detected when the enzyme reacts with its substrate, producing a detectable product. Coutlee et al. (1989) Analytical Biochemistry 181:153-162; Bogulavski (1986) et al. J. Immunol. Methods 89:123-130; Prooijen-Knegt (1982) Exp. Cell Res. 141:397-407; Rudkin (1976) Nature 265:472-473, Stollar (1970) Proc. Nat'l Acad. Sci. USA 65:993-1000; Ballard (1982) Mol. Immunol. 19:793-799; Pisetsky and Caster (1982) Mol. Immunol. 19:645-650; Viscidi et al. (1988) J. Clin. Microbial. 41:199-209; and Kiney et al. (1989) J. Clin. Microbiol. 27:6-12 describe antibodies to RNA duplexes, including homo and heteroduplexes. Kits comprising antibodies specific for DNA:RNA hybrids are available, e.g., from Digene Diagnostics, Inc. (Beltsville, MD).
  • In addition to available antibodies, one of skill in the art can easily make antibodies specific for nucleic acid duplexes using existing techniques, or modify those antibodies that are commercially or publicly available. In addition to the art referenced above, general methods for producing polyclonal and monoclonal antibodies are known to those of skill in the art (see, e.g., Paul (3rd ed.) Fundamental Immunology Raven Press, Ltd., NY (1993); Coligan Current Protocols in Immunology Wiley/Greene, NY (1991); Harlow and Lane Antibodies: A Laboratory Manual Cold Spring Harbor Press, NY (1988); Stites et al. (eds.) Basic and Clinical Immunology (4th ed.) Lange Medical Publications, Los Altos, CA, and references cited therein; Goding Monoclonal Antibodies: Principles and Practice (2d ed.) Academic Press, New York, NY, (1986); and Kohler and Milstein Nature 256: 495-497 (1975)). Other suitable techniques for antibody preparation include selection of libraries of recombinant antibodies in phage or similar vectors (see, Huse et al. Science 246:1275-1281 (1989); and Ward et al. Nature 341:544-546 (1989)). Specific monoclonal and polyclonal antibodies and antisera will usually bind with a KD of at least about 0.1 µM, preferably at least about 0.01 µM or better, and most typically and preferably, 0.001 µM or better.
  • The nucleic acids used in this invention can be either positive or negative probes. Positive probes bind to their targets and the presence of duplex formation is evidence of the presence of the target. Negative probes fail to bind to the suspect target and the absence of duplex formation is evidence of the presence of the target. For example, the use of a wild type specific nucleic acid probe or PCR primers may serve as a negative probe in an assay sample where only the nucleotide sequence of interest is present.
  • The sensitivity of the hybridization assays may be enhanced through use of a nucleic acid amplification system that multiplies the target nucleic acid being detected. Examples of such systems include the polymerase chain reaction (PCR) system, in particular RT-PCR or real time PCR, and the ligase chain reaction (LCR) system. Other methods recently described in the art are the nucleic acid sequence based amplification (NASBA, Cangene, Mississauga, Ontario) and Q Beta Replicase systems. These systems can be used to directly identify mutants where the PCR or LCR primers are designed to be extended or ligated only when a selected sequence is present. Alternatively, the selected sequences can be generally amplified using, for example, nonspecific PCR primers and the amplified target region later probed for a specific sequence indicative of a mutation.
  • An alternative means for determining the level of expression of the nucleic acids of the present invention is in situ hybridization. In situ hybridization assays are well known and are generally described in Angerer et al., Methods Enzymol. 152:649-660 (1987). In an in situ hybridization assay, cells, preferentially human cells from the cerebellum or the hippocampus, are fixed to a solid support, typically a glass slide. If DNA is to be probed, the cells are denatured with heat or alkali. The cells are then contacted with a hybridization solution at a moderate temperature to permit annealing of specific probes that are labeled. The probes are preferably labeled with radioisotopes or fluorescent reporters.
  • V. Immunological detection of the polypeptides of the invention
  • In addition to the detection of polynucleotide expression using nucleic acid hybridization technology, one can also use immunoassays to detect polypeptides of the invention. Immunoassays can be used to qualitatively or quantitatively analyze polypeptides. A general overview of the applicable technology can be found in Harlow & Lane, Antibodies: A Laboratory Manual (1988).
  • A. Antibodies to target polypeptides or other immunogens
  • Methods for producing polyclonal and monoclonal antibodies that react specifically with a protein of interest or other immunogen are known to those of skill in the art (see, e.g., Coligan, supra; and Harlow and Lane, supra; Stites et al., supra and references cited therein; Goding, supra; and Kohler and Milstein Nature, 256:495-497 (1975)). Such techniques include antibody preparation by selection of antibodies from libraries of recombinant antibodies in phage or similar vectors (see, Huse et al., supra; and Ward et al., supra). For example, in order to produce antisera for use in an immunoassay, the protein of interest or an antigenic fragment thereof, is isolated as described herein. For example, a recombinant protein is produced in a transformed cell line. An inbred strain of mice or rabbits is immunized with the protein using a standard adjuvant, such as Freund's adjuvant, and a standard immunization protocol. Alternatively, a synthetic peptide derived from the sequences disclosed herein and conjugated to a carrier protein can be used as an immunogen.
  • Polyclonal sera are collected and titered against the immunogen in an immunoassay, for example, a solid phase immunoassay with the immunogen immobilized on a solid support. Polyclonal antisera with a titer of 104 or greater are selected and tested for their cross-reactivity against unrelated proteins or even other homologous proteins from other organisms, using a competitive binding immunoassay. Specific monoclonal and polyclonal antibodies and antisera will usually bind with a KD of at least about 0.1 mM, more usually at least about 1 µM, preferably at least about 0.1 µM or better, and most preferably, 0.01 µM or better.
  • A number of proteins of the invention comprising immunogens may be used to produce antibodies specifically or selectively reactive with the proteins of interest. Recombinant protein is the preferred immunogen for the production of monoclonal or polyclonal antibodies. Naturally occurring protein, such as one comprising an amino acid sequence encoded by a gene listed in Table 1-8 may also be used either in pure or impure form. Synthetic peptides made using the protein sequences described herein may also be used as an immunogen for the production of antibodies to the protein. Recombinant protein can be expressed in eukaryotic or prokaryotic cells and purified as generally described supra. The product is then injected into an animal capable of producing antibodies. Either monoclonal or polyclonal antibodies may be generated for subsequent use in immunoassays to measure the protein.
  • Methods of production of polyclonal antibodies are known to those of skill in the art. In brief, an immunogen, preferably a purified protein, is mixed with an adjuvant and animals are immunized. The animal's immune response to the immunogen preparation is monitored by taking test bleeds and determining the titer of reactivity to the polypeptide of interest. When appropriately high titers of antibody to the immunogen are obtained, blood is collected from the animal and antisera are prepared. Further fractionation of the antisera to enrich for antibodies reactive to the protein can be done if desired (see, Harlow and Lane, supra).
  • Monoclonal antibodies may be obtained using various techniques familiar to those of skill in the art. Typically, spleen cells from an animal immunized with a desired antigen are immortalized, commonly by fusion with a myeloma cell (see, Kohler and Milstein, Eur. J. Immunol. 6:511-519 (1976)). Alternative methods of immortalization include, e.g., transformation with Epstein Barr Virus, oncogenes, or retroviruses, or other methods well known in the art. Colonies arising from single immortalized cells are screened for production of antibodies of the desired specificity and affinity for the antigen, and yield of the monoclonal antibodies produced by such cells may be enhanced by various techniques, including injection into the peritoneal cavity of a vertebrate host. Alternatively, one may isolate DNA sequences which encode a monoclonal antibody or a binding fragment thereof by screening a DNA library from human B cells according to the general protocol outlined by Huse et al., supra.
  • Once target protein specific antibodies are available, the protein can be measured by a variety of immunoassay methods with qualitative and quantitative results available to the clinician. For a review of immunological and immunoassay procedures in general see, Stites, supra. Moreover, the immunoassays of the present invention can be performed in any of several configurations, which are reviewed extensively in Maggio Enzyme Immunoassay, CRC Press, Boca Raton, Florida (1980); Tijssen, supra; and Harlow and Lane, supra.
  • Immunoassays to measure target proteins in a human sample may use a polyclonal antiserum that was raised to the protein (e.g., one has an amino acid sequence encoded by a gene listed in Table 1-8) or a fragment thereof. This antiserum is selected to have low cross-reactivity against different proteins and any such cross-reactivity is removed by immunoabsorption prior to use in the immunoassay.
  • B. Immunological Binding Assays
  • In a preferred embodiment, a protein of interest is detected and/or quantified using any of a number of well-known immunological binding assays (see, e.g., U.S. Patents 4,366,241 ; 4,376,110 ; 4,517,288 ; and 4,837,168 ). For a review of the general immunoassays, see also Asai Methods in Cell Biology Volume 37: Antibodies in Cell Biology, Academic Press, Inc. NY (1993); Stites, supra. Immunological binding assays (or immunoassays) typically utilize a "capture agent" to specifically bind to and often immobilize the analyte (in this case a polypeptide of the present invention or antigenic subsequences thereof). The capture agent is a moiety that specifically binds to the analyte. In a preferred embodiment, the capture agent is an antibody that specifically binds, for example, a polypeptide of the invention. The antibody may be produced by any of a number of means well known to those of skill in the art and as described above.
  • Immunoassays also often utilize a labeling agent to specifically bind to and label the binding complex formed by the capture agent and the analyte. The labeling agent may itself be one of the moieties comprising the antibody/analyte complex. Alternatively, the labeling agent may be a third moiety, such as another antibody, that specifically binds to the antibody/protein complex.
  • In a preferred embodiment, the labeling agent is a second antibody bearing a label. Alternatively, the second antibody may lack a label, but it may, in turn, be bound by a labeled third antibody specific to antibodies of the species from which the second antibody is derived. The second antibody can be modified with a detectable moiety, such as biotin, to which a third labeled molecule can specifically bind, such as enzyme-labeled streptavidin.
  • Other proteins capable of specifically binding immunoglobulin constant regions, such as protein A or protein G, can also be used as the label agents. These proteins are normal constituents of the cell walls of streptococcal bacteria. They exhibit a strong non-immunogenic reactivity with immunoglobulin constant regions from a variety of species (see, generally, Kronval, et al. J. Immunol., 111:1401-1406 (1973); and Akerstrom, et al. J. Immunol., 135:2589-2542 (1985)).
  • Throughout the assays, incubation and/or washing steps may be required after each combination of reagents. Incubation steps can vary from about 5 seconds to several hours, preferably from about 5 minutes to about 24 hours. The incubation time will depend upon the assay format, analyte, volume of solution, concentrations, and the like. Usually, the assays will be carried out at ambient temperature, although they can be conducted over a range of temperatures, such as 10°C to 40°C.
  • 1. Non-Competitive Assay Formats
  • Immunoassays for detecting proteins of interest from tissue samples may be either competitive or noncompetitive. Noncompetitive immunoassays are assays in which the amount of captured analyte (in this case the protein) is directly measured. In one preferred "sandwich" assay, for example, the capture agent (e.g., antibodies specific for a polypeptide encoded by a gene listed in Table 1-8) can be bound directly to a solid substrate where it is immobilized. These immobilized antibodies then capture the polypeptide present in the test sample. The polypeptide thus immobilized is then bound by a labeling agent, such as a second antibody bearing a label. Alternatively, the second antibody may lack a label, but it may, in turn, be bound by a labeled third antibody specific to antibodies of the species from which the second antibody is derived. The second can be modified with a detectable moiety, such as biotin, to which a third labeled molecule can specifically bind, such as enzyme-labeled streptavidin.
  • 2. Competitive Assay Formats
  • In competitive assays, the amount of analyte (such as a polypeptide encoded by a gene listed in Table 1-8) present in the sample is measured indirectly by measuring the amount of an added (exogenous) analyte displaced (or competed away) from a capture agent (e.g., an antibody specific for the analyte) by the analyte present in the sample. In one competitive assay, a known amount of, in this case, the protein of interest is added to the sample and the sample is then contacted with a capture agent, in this case an antibody that specifically binds to a polypeptide of the invention. The amount of immunogen bound to the antibody is inversely proportional to the concentration of immunogen present in the sample. In a particularly preferred embodiment, the antibody is immobilized on a solid substrate. For example, the amount of the polypeptide bound to the antibody may be determined either by measuring the amount of subject protein present in a protein/antibody complex or, alternatively, by measuring the amount of remaining uncomplexed protein. The amount of protein may be detected by providing a labeled protein molecule.
  • Immunoassays in the competitive binding format can be used for cross-reactivity determinations. For example, a protein of interest can be immobilized on a solid support. Proteins are added to the assay which compete with the binding of the antisera to the immobilized antigen. The ability of the above proteins to compete with the binding of the antisera to the immobilized protein is compared to that of the protein of interest. The percent cross-reactivity for the above proteins is calculated, using standard calculations. Those antisera with less than 10% cross-reactivity with each of the proteins listed above are selected and pooled. The cross-reacting antibodies are optionally removed from the pooled antisera by immunoabsorption with the considered proteins, e.g., distantly related homologs.
  • The immunoabsorbed and pooled antisera are then used in a competitive binding immunoassay as described above to compare a second protein, thought to be perhaps a protein of the present invention, to the immunogen protein. In order to make this comparison, the two proteins are each assayed at a wide range of concentrations and the amount of each protein required to inhibit 50% of the binding of the antisera to the immobilized protein is determined. If the amount of the second protein required is less than 10 times the amount of the protein partially encoded by a sequence herein that is required, then the second protein is said to specifically bind to an antibody generated to an immunogen consisting of the target protein.
  • 3. Other Assay Formats
  • In a particularly preferred embodiment, western blot (immunoblot) analysis is used to detect and quantify the presence of a polypeptide of the invention in the sample. The technique generally comprises separating sample proteins by gel electrophoresis on the basis of molecular weight, transferring the separated proteins to a suitable solid support (such as, e.g., a nitrocellulose filter, a nylon filter, or a derivatized nylon filter) and incubating the sample with the antibodies that specifically bind the protein of interest. For example, the antibodies specifically bind to a polypeptide of interest on the solid support. These antibodies may be directly labeled or alternatively may be subsequently detected using labeled antibodies (e.g., labeled sheep anti-mouse antibodies) that specifically bind to the antibodies against the protein of interest.
  • Other assay formats include liposome immunoassays (LIA), which use liposomes designed to bind specific molecules (e.g., antibodies) and release encapsulated reagents or markers. The released chemicals are then detected according to standard techniques (see, Monroe et al. (1986) Amer. Clin. Prod. Rev. 5:34-41).
  • 4. Labels
  • The particular label or detectable group used in the assay is not a critical aspect of the invention, as long as it does not significantly interfere with the specific binding of the antibody used in the assay. The detectable group can be any material having a detectable physical or chemical property. Such detectable labels have been well developed in the field of immunoassays and, in general, most labels useful in such methods can be applied to the present invention. Thus, a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Useful labels in the present invention include magnetic beads (e.g., Dynabeads), fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the like), radiolabels (e.g., 3H, 125I, 35S, 14C, or 32P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads.
  • The label may be coupled directly or indirectly to the desired component of the assay according to methods well known in the art. As indicated above, a wide variety of labels may be used, with the choice of label depending on the sensitivity required, the ease of conjugation with the compound, stability requirements, available instrumentation, and disposal provisions.
  • Non-radioactive labels are often attached by indirect means. The molecules can also be conjugated directly to signal generating compounds, e.g., by conjugation with an enzyme or fluorescent compound. A variety of enzymes and fluorescent compounds can be used with the methods of the present invention and are well-known to those of skill in the art (for a review of various labeling or signal producing systems which may be used, see, e.g., U.S. Patent No. 4,391,904 ).
  • Means of detecting labels are well known to those of skill in the art. Thus, for example, where the label is a radioactive label, means for detection include a scintillation counter or photographic film as in autoradiography. Where the label is a fluorescent label, it may be detected by exciting the fluorochrome with the appropriate wavelength of light and detecting the resulting fluorescence. The fluorescence may be detected visually, by means of photographic film, by the use of electronic detectors such as charge-coupled devices (CCDs) or photomultipliers and the like. Similarly, enzymatic labels may be detected by providing the appropriate substrates for the enzyme and detecting the resulting reaction product. Finally simple colorimetric labels may be detected directly by observing the color associated with the label. Thus, in various dipstick assays, conjugated gold often appears pink, while various conjugated beads appear the color of the bead.
  • Some assay formats do not require the use of labeled components. For instance, agglutination assays can be used to detect the presence of the target antibodies. In this case, antigen-coated particles are agglutinated by samples comprising the target antibodies. In this format, none of the components need to be labeled and the presence of the target antibody is detected by simple visual inspection.
  • In some embodiments, BP or MDD in a patient may be diagnosed or otherwise evaluated by visualizing expression in situ of one or more of the appropriately dysregulated gene sequences identified herein. Those skilled in the art of visualizing the presence or expression of molecules including nucleic acids, polypeptides and other biochemicals in the brains of living patients will appreciate that the gene expression information described herein may be utilized in the context of a variety of visualization methods. Such methods include, but are not limited to, single-photon emission-computed tomography (SPECT) and positron-emitting tomography (PET) methods. See, e.g., Vassaux and Groot-wassink, "In Vivo Noninvasive Imaging for Gene Therapy," J. Biomedicine and Biotechnology, 2: 92-101 (2003).
  • PET and SPECT imaging shows the chemical functioning of organs and tissues, while other imaging techniques - such as X-ray, CT and MRI - show structure. The use of PET and SPECT imaging is useful for qualifying and monitoring the development of brain diseases, including schizophrenia and related disorders. In some instances, the use of PET or SPECT imaging allows diseases to be detected years earlier than the onset of symptoms. The use of small molecules for labelling and visualizing the presence or expression of polypeptides and nucleotides has had success, for example, in visualizing proteins in the brains of Alzheimer's patients, as described by, e.g., Herholz K et al., Mol Imaging Biol., 6(4):239-69 (2004); Nordberg A, Lancet Neurol., 3(9):519-27 (2004); Neuropsychol Rev., Zakzanis KK et al., 13(1):1-18 (2003); Kung MP et al, Brain Res.,1025(1-2):98-105 (2004); and Herholz K, Ann Nucl Med., 17(2):79-89 (2003).
  • The dysregulated genes disclosed in Tables 1-30, or their encoded peptides (if any), or fragments thereof, can be used in the context of PET and SPECT imaging applications. After modification with appropriate tracer residues for PET or SPECT applications, molecules which interact or bind with the transcripts in Tables 1-30 or with any polypeptides encoded by those transcripts may be used to visualize the patterns of gene expression and facilitate diagnosis of schizophrenia MDD or BP, as described herein. Similarly, if the encoded polypeptides encode enzymes, labeled molecules which interact with the products of catalysis by the enzyme may be used for the in vivo imaging and diagnostic application described herein.
  • Antisense technology is particularly suitable for detecting the the transcripts identified in Tables 1-30 herein. For example, the use of antisense peptide nucleic acid (PNA) labeled with an appropriate radionuclide, such as 111In, and conjugated to a brain drug-targeting system to enable transport across biologic membrane barriers, has been demonstrated to allow imaging of endogenous gene expression in brain cancer. See Suzuki et al., Journal of Nuclear Medicine, 10: 1766-1775 (2004). Suzuki et al. utilize a delivery system comprising monoclonal antibodies that target transferring receptors at the blood-brain barrier and facilitate transport of the PNA across that barrier. Modified embodiments of this technique may be used to target upregulated genes associated with schizophrenia, BP or MDD, such as the upregulated genes which appear in Tables 1-30, in methods of treating schizophrenic, BP or MDD patients.
  • In other embodiments, the dysregulated genes listed in Tables 1-30 may be used in the context of prenatal and neonatal diagnostic methods. For example, fetal or neonatal samples can be obtained and the expression levels of appropriate transcripts (e.g., the transcripts in Table 19) may be measured and correlated with the presence or increased likelihood of a mental disorder, e.g., MDD. Similarly, the presence of one or more of the SNPs identified in the Tables, e.g., Table 27 may be used to infer or corroborate dysregulated expression of a gene and the likelihood of a mood disorder in prenatal, neonatal, children and adult patients.
  • In other embodiments, the brain labeling and imaging techniques described herein or variants thereof may be used in conjunction with any of the dysregulated gene sequences in Tables 1-30 in a forensic analysis, i.e., to determine whether a deceased individual suffered from schizophrenia, BP, or MDD.
  • VI. Screening for modulators of polypeptides and polynucleotides of the invention
  • Modulators of polypeptides or polynucleotides of the invention, i.e. agonists or antagonists of their activity or modulators of polypeptide or polynucleotide expression, are useful for treating a number of human diseases, including mood disorders or psychotic disorders. Administration of agonists, antagonists or other agents that modulate expression of the polynucleotides or polypeptides of the invention can be used to treat patients with mood disorders or psychotic disorders.
  • A. Screening methods
  • A number of different screening protocols can be utilized to identify agents that modulate the level of expression or activity of polypeptides and polynucleotides of the invention in cells, particularly mammalian cells, and especially human cells. In general terms, the screening methods involve screening a plurality of agents to identify an agent that modulates the polypeptide activity by binding to a polypeptide of the invention, modulating inhibitor binding to the polypeptide or activating expression of the polypeptide or polynucleotide, for example.
  • 1. Binding Assays
  • Preliminary screens can be conducted by screening for agents capable of binding to a polypeptide of the invention, as at least some of the agents so identified are likely modulators of polypeptide activity. The binding assays usually involve contacting a polypeptide of the invention with one or more test agents and allowing sufficient time for the protein and test agents to form a binding complex. Any binding complexes formed can be detected using any of a number of established analytical techniques. Protein binding assays include, but are not limited to, methods that measure co-precipitation, co-migration on non-denaturing SDS-polyacrylamide gels, and co-migration on Western blots (see, e.g., Bennet and Yamamura, (1985) "Neurotransmitter, Hormone or Drug Receptor Binding Methods," in Neurotransmitter Receptor Binding (Yamamura, H. I., et al., eds.), pp. 61-89. The protein utilized in such assays can be naturally expressed, cloned or synthesized.
  • Binding assays are also useful, e.g., for identifying endogenous proteins that interact with a polypeptide of the invention. For example, antibodies, receptors or other molecules that bind a polypeptide of the invention can be identified in binding assays.
  • 2. Expression Assays
  • Certain screening methods involve screening for a compound that up or down-regulates the expression of a polypeptide or polynucleotide of the invention. Such methods generally involve conducting cell-based assays in which test compounds are contacted with one or more cells expressing a polypeptide or polynucleotide of the invention and then detecting an increase or decrease in expression (either transcript, translation product, or catalytic product). Some assays are performed with peripheral cells, or other cells, that express an endogenous polypeptide or polynucleotide of the invention.
  • Polypeptide or polynucleotide expression can be detected in a number of different ways. As described infra, the expression level of a polynucleotide of the invention in a cell can be determined by probing the mRNA expressed in a cell with a probe that specifically hybridizes with a transcript (or complementary nucleic acid derived therefrom) of a polynucleotide of the invention. Probing can be conducted by lysing the cells and conducting Northern blots or without lysing the cells using in situ-hybridization techniques. Alternatively, a polypeptide of the invention can be detected using immunological methods in which a cell lysate is probed with antibodies that specifically bind to a polypeptide of the invention.
  • Other cell-based assays are reporter assays conducted with cells that do not express a polypeptide or polynucleotide of the invention. Certain of these assays are conducted with a heterologous nucleic acid construct that includes a promoter of a polynucleotide of the invention that is operably linked to a reporter gene that encodes a detectable product. A number of different reporter genes can be utilized. Some reporters are inherently detectable. An example of such a reporter is green fluorescent protein that emits fluorescence that can be detected with a fluorescence detector. Other reporters generate a detectable product. Often such reporters are enzymes. Exemplary enzyme reporters include, but are not limited to, β-glucuronidase, chloramphenicol acetyl transferase (CAT); Alton and Vapnek (1979) Nature 282:864-869), luciferase, β-galactosidase, green fluorescent protein (GFP) and alkaline phosphatase (Toh, et al. (1980) Eur. J. Biochem. 182:231-238; and Hall et al. (1983) J. Mol. Appl. Gen. 2:101).
  • In these assays, cells harboring the reporter construct are contacted with a test compound. A test compound that either activates the promoter by binding to it or triggers a cascade that produces a molecule that activates the promoter causes expression of the detectable reporter. Certain other reporter assays are conducted with cells that harbor a heterologous construct that includes a transcriptional control element that activates expression of a polynucleotide of the invention and a reporter operably linked thereto. Here, too, an agent that binds to the transcriptional control element to activate expression of the reporter or that triggers the formation of an agent that binds to the transcriptional control element to activate reporter expression, can be identified by the generation of signal associated with reporter expression.
  • The level of expression or activity can be compared to a baseline value. As indicated above, the baseline value can be a value for a control sample or a statistical value that is representative of expression levels for a control population (e.g., healthy individuals not having or at risk for mood disorders or psychotic disorders). Expression levels can also be determined for cells that do not express a polynucleotide of the invention as a negative control. Such cells generally are otherwise substantially genetically the same as the test cells.
  • A variety of different types of cells can be utilized in the reporter assays. Cells that express an endogenous polypeptide or polynucleotide of the invention include, e.g., brain cells, including cells from the cerebellum, anterior cingulate cortex, dorsolateral prefrontal cortex, amygdala, hippocampus, or nucleus accumbens. Cells that do not endogenously express polynucleotides of the invention can be prokaryotic, but are preferably eukaryotic. The eukaryotic cells can be any of the cells typically utilized in generating cells that harbor recombinant nucleic acid constructs. Exemplary eukaryotic cells include, but are not limited to, yeast, and various higher eukaryotic cells such as the COS, CHO and HeLa cell lines.
  • Various controls can be conducted to ensure that an observed activity is authentic including running parallel reactions with cells that lack the reporter construct or by not contacting a cell harboring the reporter construct with test compound. Compounds can also be further validated as described below.
  • 3. Catalytic activity
  • Catalytic activity of polypeptides of the invention can be determined by measuring the production of enzymatic products or by measuring the consumption of substrates. Activity refers to either the rate of catalysis or the ability to the polypeptide to bind (Km) the substrate or release the catalytic product (Kd).
  • Analysis of the activity of polypeptides of the invention are performed according to general biochemical analyses. Such assays include cell-based assays as well as in vitro assays involving purified or partially purified polypeptides or crude cell lysates. The assays generally involve providing a known quantity of substrate and quantifying product as a function of time.
  • 4. Validation
  • Agents that are initially identified by any of the foregoing screening methods can be further tested to validate the apparent activity. Preferably such studies are conducted with suitable animal models. The basic format of such methods involves administering a lead compound identified during an initial screen to an animal that serves as a model for humans and then determining if expression or activity of a polynucleotide or polypeptide of the invention is in fact upregulated. The animal models utilized in validation studies generally are mammals of any kind. Specific examples of suitable animals include, but are not limited to, primates, mice, and rats. As described herein, models using admininstration of known therapeutics can be useful.
  • 5. Animal models
  • Animal models of mental disorders also find use in screening for modulators. In one embodiment, invertebrate models such as Drosophila models can be used, screening for modulators of Drosophila orthologs of the human genes disclosed herein. In another embodiment, transgenic animal technology including gene knockout technology, for example as a result of homologous recombination with an appropriate gene targeting vector, or gene overexpression, will result in the absence, decreased or increased expression of a polynucleotide or polypeptide of the invention. The same technology can also be applied to make knockout cells. When desired, tissue-specific expression or knockout of a polynucleotide or polypeptide of the invention may be necessary. Transgenic animals generated by such methods find use as animal models of mental illness and are useful in screening for modulators of mental illness.
  • Knockout cells and transgenic mice can be made by insertion of a marker gene or other heterologous gene into an endogenous gene site in the mouse genome via homologous recombination. Such mice can also be made by substituting an endogenous polynucleotide of the invention with a mutated version of the polynucleotide, or by mutating an endogenous polynucleotide, e.g., by exposure to carcinogens.
  • For development of appropriate stem cells, a DNA construct is introduced into the nuclei of embryonic stem cells. Cells containing the newly engineered genetic lesion are injected into a host mouse embryo, which is re-implanted into a recipient female. Some of these embryos develop into chimeric mice that possess germ cells partially derived from the mutant cell line. Therefore, by breeding the chimeric mice it is possible to obtain a new line of mice containing the introduced genetic lesion (see, e.g., Capecchi et al., Science 244:1288 (1989)). Chimeric targeted mice can be derived according to Hogan et al., Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory (1988) and Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, Robertson, ed., IRL Press, Washington, D.C., (1987).
  • B. Modulators of polypeptides or polynucleotides of the invention
  • The agents tested as modulators of the polypeptides or polynucleotides of the invention can be any small chemical compound, or a biological entity, such as a protein, sugar, nucleic acid or lipid. Alternatively, modulators can be genetically altered versions of a polypeptide or polynucleotide of the invention. Typically, test compounds will be small chemical molecules and peptides. Essentially any chemical compound can be used as a potential modulator or ligand in the assays of the invention, although most often compounds that can be dissolved in aqueous or organic (especially DMSO-based) solutions are used. The assays are designed to screen large chemical libraries by automating the assay steps and providing compounds from any convenient source to assays, which are typically run in parallel (e.g., in microtiter formats on microtiter plates in robotic assays). It will be appreciated that there are many suppliers of chemical compounds, including Sigma (St. Louis, MO), Aldrich (St. Louis, MO), Sigma-Aldrich (St. Louis, MO), Fluka Chemika-Biochemica Analytika (Buchs, Switzerland) and the like. Modulators also include agents designed to reduce the level of mRNA of the invention (e.g. antisense molecules, ribozymes, DNAzymes and the like) or the level of translation from an mRNA.
  • In one preferred embodiment, high throughput screening methods involve providing a combinatorial chemical or peptide library containing a large number of potential therapeutic compounds (potential modulator or ligand compounds). Such "combinatorial chemical libraries" or "ligand libraries" are then screened in one or more assays, as described herein, to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity. The compounds thus identified can serve as conventional "lead compounds" or can themselves be used as potential or actual therapeutics.
  • A combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis, by combining a number of chemical "building blocks" such as reagents. For example, a linear combinatorial chemical library such as a polypeptide library is formed by combining a set of chemical building blocks (amino acids) in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks.
  • Preparation and screening of combinatorial chemical libraries is well known to those of skill in the art. Such combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Patent 5,010,175 , Furka, Int. J. Pept. Prot. Res. 37:487-493 (1991) and Houghton et al., Nature 354:84-88 (1991)). Other chemistries for generating chemical diversity libraries can also be used. Such chemistries include, but are not limited to: peptoids (e.g., PCT Publication No. WO 91/19735 ), encoded peptides (e.g., PCT Publication WO 93/20242 ), random bio-oligomers (e.g., PCT Publication No. WO 92/00091 ), benzodiazepines (e.g., U.S. Pat. No. 5,288,514 ), diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc. Nat. Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypeptides (Hagihara et al., J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidal peptidomimetics with glucose scaffolding (Hirschmann et al., J. Amer. Chem. Soc. 114:9217-9218 (1992)), analogous organic syntheses of small compound libraries (Chen et al., J. Amer. Chem. Soc. 116:2661 (1994)), oligocarbamates (Cho et al., Science 261:1303 (1993)), and/or peptidyl phosphonates (Campbell et al., J. Org. Chem. 59:658 (1994)), nucleic acid libraries (see Ausubel, Berger and Sambrook, all supra), peptide nucleic acid libraries (see, e.g., U.S. Patent 5,539,083 ), antibody libraries (see, e.g., Vaughn et al., Nature Biotechnology, 14(3):309-314 (1996) and PCT/US96/10287 ), carbohydrate libraries (see, e.g., Liang et al., Science, 274:1520-1522 (1996) and U.S. Patent 5,593,853 ), small organic molecule libraries (see, e.g., benzodiazepines, Baum C&EN, ); isoprenoids, U.S. Patent 5,569,588 ; thiazolidinones and metathiazanones, U.S. Patent 5,549,974 ; pyrrolidines, U.S. Patents 5,525,735 and 5,519,134 ; morpholino compounds, U.S. Patent 5,506,337 ; benzodiazepines, 5,288,514 , and the like).
  • Devices for the preparation of combinatorial libraries are commercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, Louisville KY; Symphony, Rainin, Woburn, MA; 433A Applied Biosystems, Foster City, CA; 9050 Plus, Millipore, Bedford, MA). In addition, numerous combinatorial libraries are themselves commercially available (see, e.g., ComGenex, Princeton, NJ; Tripos, Inc., St. Louis, MO; 3D Pharmaceuticals, Exton, PA; Martek Biosciences, Columbia, MD, etc.).
  • C. Solid State and Soluble High Throughput Assays
  • In the high throughput assays of the invention, it is possible to screen up to several thousand different modulators or ligands in a single day. In particular, each well of a microtiter plate can be used to run a separate assay against a selected potential modulator, or, if concentration or incubation time effects are to be observed, every 5-10 wells can test a single modulator. Thus, a single standard microtiter plate can assay about 100 (e.g., 96) modulators. If 1536 well plates are used, then a single plate can easily assay from about 100 to about 1500 different compounds. It is possible to assay several different plates per day; assay screens for up to about 6,000-20,000 different compounds are possible using the integrated systems of the invention. More recently, microfluidic approaches to reagent manipulation have been developed.
  • The molecule of interest can be bound to the solid state component, directly or indirectly, via covalent or non-covalent linkage, e.g., via a tag. The tag can be any of a variety of components. In general, a molecule that binds the tag (a tag binder) is fixed to a solid support, and the tagged molecule of interest is attached to the solid support by interaction of the tag and the tag binder.
  • A number of tags and tag binders can be used, based upon known molecular interactions well described in the literature. For example, where a tag has a natural binder, for example, biotin, protein A, or protein G, it can be used in conjunction with appropriate tag binders (avidin, streptavidin, neutravidin, the Fc region of an immunoglobulin, etc.). Antibodies to molecules with natural binders such as biotin are also widely available and appropriate tag binders (see, SIGMA Immunochemicals 1998 catalogue SIGMA, St. Louis MO).
  • Similarly, any haptenic or antigenic compound can be used in combination with an appropriate antibody to form a tag/tag binder pair. Thousands of specific antibodies are commercially available and many additional antibodies are described in the literature. For example, in one common configuration, the tag is a first antibody and the tag binder is a second antibody which recognizes the first antibody. In addition to antibody-antigen interactions, receptor-ligand interactions are also appropriate as tag and tag-binder pairs, such as agonists and antagonists of cell membrane receptors (e.g., cell receptor-ligand interactions such as transferrin, c-kit, viral receptor ligands, cytokine receptors, chemokine receptors, interleukin receptors, immunoglobulin receptors and antibodies, the cadherin family, the integrin family, the selectin family, and the like; see, e.g., Pigott & Power, The Adhesion Molecule Facts Book I (1993)). Similarly, toxins and venoms, viral epitopes, hormones (e.g., opiates, steroids, etc.), intracellular receptors (e.g., which mediate the effects of various small ligands, including steroids, thyroid hormone, retinoids and vitamin D; peptides), drugs, lectins, sugars, nucleic acids (both linear and cyclic polymer configurations), oligosaccharides, proteins, phospholipids and antibodies can all interact with various cell receptors.
  • Synthetic polymers, such as polyurethanes, polyesters, polycarbonates, polyureas, polyamides, polyethyleneimines, polyarylene sulfides, polysiloxanes, polyimides, and polyacetates can also form an appropriate tag or tag binder. Many other tag/tag binder pairs are also useful in assay systems described herein, as would be apparent to one of skill upon review of this disclosure.
  • Common linkers such as peptides, polyethers, and the like can also serve as tags, and include polypeptide sequences, such as poly-Gly sequences of between about 5 and 200 amino acids. Such flexible linkers are known to those of skill in the art. For example, poly(ethelyne glycol) linkers are available from Shearwater Polymers, Inc., Huntsville, Alabama. These linkers optionally have amide linkages, sulfhydryl linkages, or heterofunctional linkages.
  • Tag binders are fixed to solid substrates using any of a variety of methods currently available. Solid substrates are commonly derivatized or functionalized by exposing all or a portion of the substrate to a chemical reagent which fixes a chemical group to the surface which is reactive with a portion of the tag binder. For example, groups which are suitable for attachment to a longer chain portion would include amines, hydroxyl, thiol, and carboxyl groups. Aminoalkylsilanes and hydroxyalkylsilanes can be used to functionalize a variety of surfaces, such as glass surface. The construction of such solid phase biopolymer arrays is well described in the literature (see, e.g., Merrifield, J. Am. Chem. Soc. 85:2149-2154 (1963) (describing solid phase synthesis of, e.g., peptides); Geysen et al., J. Immun. Meth. 102:259-274 (1987) (describing synthesis of solid phase components on pins); Frank and Doring, Tetrahedron 44:60316040 (1988) (describing synthesis of various peptide sequences on cellulose disks); Fodor et al., Science, 251:767-777 (1991); Sheldon et al., Clinical Chemistry 39(4):718-719 (1993); and Kozal et al., Nature Medicine 2(7):753759(1996) (all describing arrays of biopolymers fixed to solid substrates). Non-chemical approaches for fixing tag binders to substrates include other common methods, such as heat, cross-linking by UV radiation, and the like.
  • The invention provides in vitro assays for identifying, in a high throughput format, compounds that can modulate the expression or activity of the polynucleotides or polypeptides of the invention. In a preferred embodiment, the methods of the invention include such a control reaction. For each of the assay formats described, "no modulator" control reactions that do not include a modulator provide a background level of binding activity.
  • In some assays it will be desirable to have positive controls to ensure that the components of the assays are working properly. At least two types of positive controls are appropriate. First, a known activator of a polynucleotide or polypeptide of the invention can be incubated with one sample of the assay, and the resulting increase in signal resulting from an increased expression level or activity of polynucleotide or polypeptide determined according to the methods herein. Second, a known inhibitor of a polynucleotide or polypeptide of the invention can be added, and the resulting decrease in signal for the expression or activity can be similarly detected.
  • D. Computer-Based Assays
  • Yet another assay for compounds that modulate the activity of a polypeptide or polynucleotide of the invention involves computer assisted drug design, in which a computer system is used to generate a three-dimensional structure of the polypeptide or polynucleotide based on the structural information encoded by its amino acid or nucleotide sequence. The input sequence interacts directly and actively with a pre-established algorithm in a computer program to yield secondary, tertiary, and quaternary structural models of the molecule. Similar analyses can be performed on potential receptors or binding partners of the polypeptides or polynucleotides of the invention. The models of the protein or nucleotide structure are then examined to identify regions of the structure that have the ability to bind, e.g., a polypeptide or polynucleotide of the invention. These regions are then used to identify polypeptides that bind to a polypeptide or polynucleotide of the invention.
  • The three-dimensional structural model of a protein is generated by entering protein amino acid sequences of at least 10 amino acid residues or corresponding nucleic acid sequences encoding a potential receptor into the computer system. The amino acid sequences encoded by the nucleic acid sequences provided herein represent the primary sequences or subsequences of the proteins, which encode the structural information of the proteins. At least 10 residues of an amino acid sequence (or a nucleotide sequence encoding 10 amino acids) are entered into the computer system from computer keyboards, computer readable substrates that include, but are not limited to, electronic storage media (e.g., magnetic diskettes, tapes, cartridges, and chips), optical media (e.g., CD ROM), information distributed by internet sites, and by RAM. The three-dimensional structural model of the protein is then generated by the interaction of the amino acid sequence and the computer system, using software known to those of skill in the art.
  • The amino acid sequence represents a primary structure that encodes the information necessary to form the secondary, tertiary, and quaternary structure of the protein of interest. The software looks at certain parameters encoded by the primary sequence to generate the structural model. These parameters are referred to as "energy terms," and primarily include electrostatic potentials, hydrophobic potentials, solvent accessible surfaces, and hydrogen bonding. Secondary energy terms include van der Waals potentials. Biological molecules form the structures that minimize the energy terms in a cumulative fashion. The computer program is therefore using these terms encoded by the primary structure or amino acid sequence to create the secondary structural model.
  • The tertiary structure of the protein encoded by the secondary structure is then formed on the basis of the energy terms of the secondary structure. The user at this point can enter additional variables such as whether the protein is membrane bound or soluble, its location in the body, and its cellular location, e.g., cytoplasmic, surface, or nuclear. These variables along with the energy terms of the secondary structure are used to form the model of the tertiary structure. In modeling the tertiary structure, the computer program matches hydrophobic faces of secondary structure with like, and hydrophilic faces of secondary structure with like.
  • Once the structure has been generated, potential ligand binding regions are identified by the computer system. Three-dimensional structures for potential ligands are generated by entering amino acid or nucleotide sequences or chemical formulas of compounds, as described above. The three-dimensional structure of the potential ligand is then compared to that of a polypeptide or polynucleotide of the invention to identify binding sites of the polypeptide or polynucleotide of the invention. Binding affinity between the protein and ligands is determined using energy terms to determine which ligands have an enhanced probability of binding to the protein.
  • Computer systems are also used to screen for mutations, polymorphic variants, alleles and interspecies homologs of genes encoding a polypeptide or polynucleotide of the invention. Such mutations can be associated with disease states or genetic traits and can be used for diagnosis. As described above, GeneChip™ and related technology can also be used to screen for mutations, polymorphic variants, alleles and interspecies homologs. Once the variants are identified, diagnostic assays can be used to identify patients having such mutated genes. Identification of the mutated a polypeptide or polynucleotide of the invention involves receiving input of a first amino acid sequence of a polypeptide of the invention (or of a first nucleic acid sequence encoding a polypeptide of the invention), e.g., any amino acid sequence having at least 60%, optionally at least 70% or 85%, identity with the amino acid sequence of interest, or conservatively modified versions thereof. The sequence is entered into the computer system as described above. The first nucleic acid or amino acid sequence is then compared to a second nucleic acid or amino acid sequence that has substantial identity to the first sequence. The second sequence is entered into the computer system in the manner described above. Once the first and second sequences are compared, nucleotide or amino acid differences between the sequences are identified. Such sequences can represent allelic differences in various polynucleotides of the invention, and mutations associated with disease states and genetic traits.
  • VII. Compositions, Kits and Integrated Systems
  • The invention provides compositions, kits and integrated systems for practicing the assays described herein using polypeptides or polynucleotides of the invention, antibodies specific for polypeptides or polynucleotides of the invention, etc.
  • The invention provides assay compositions for use in solid phase assays; such compositions can include, for example, one or more polynucleotides or polypeptides of the invention immobilized on a solid support, and a labeling reagent. In each case, the assay compositions can also include additional reagents that are desirable for hybridization. Modulators of expression or activity of polynucleotides or polypeptides of the invention can also be included in the assay compositions.
  • The invention also provides kits for carrying out the therapeutic and diagnostic assays of the invention. The kits typically include a probe that comprises an antibody that specifically binds to polypeptides or polynucleotides of the invention, and a label for detecting the presence of the probe. The kits may include several polynucleotide sequences encoding polypeptides of the invention. Kits can include any of the compositions noted above, and optionally further include additional components such as instructions to practice a high-throughput method of assaying for an effect on expression of the genes encoding the polypeptides of the invention, or on activity of the polypeptides of the invention, one or more containers or compartments (e.g., to hold the probe, labels, or the like), a control modulator of the expression or activity of polypeptides of the invention, a robotic armature for mixing kit components or the like.
  • The invention also provides integrated systems for high-throughput screening of potential modulators for an effect on the expression or activity of the polypeptides of the invention. The systems typically include a robotic armature which transfers fluid from a source to a destination, a controller which controls the robotic armature, a label detector, a data storage unit which records label detection, and an assay component such as a microtiter dish comprising a well having a reaction mixture or a substrate comprising a fixed nucleic acid or immobilization moiety.
  • A number of robotic fluid transfer systems are available, or can easily be made from existing components. For example, a Zymate XP (Zymark Corporation; Hopkinton, MA) automated robot using a Microlab 2200 (Hamilton; Reno, NV) pipetting station can be used to transfer parallel samples to 96 well microtiter plates to set up several parallel simultaneous STAT binding assays.
  • Optical images viewed (and, optionally, recorded) by a camera or other recording device (e.g., a photodiode and data storage device) are optionally further processed in any of the embodiments herein, e.g., by digitizing the image and storing and analyzing the image on a computer. A variety of commercially available peripheral equipment and software is available for digitizing, storing and analyzing a digitized video or digitized optical image, e.g., using PC (Intel x86 or Pentium chip-compatible DOS®, OS2® WINDOWS®, WINDOWS NT®, WINDOWS95®, WINDOWS98®, or WINDOWS2000® based computers), MACINTOSH®, or UNIX® based (e.g., SUN® work station) computers.
  • One conventional system carries light from the specimen field to a cooled charge-coupled device (CCD) camera, in common use in the art. A CCD camera includes an array of picture elements (pixels). The light from the specimen is imaged on the CCD. Particular pixels corresponding to regions of the specimen (e.g., individual hybridization sites on an array of biological polymers) are sampled to obtain light intensity readings for each position. Multiple pixels are processed in parallel to increase speed. The apparatus and methods of the invention are easily used for viewing any sample, e.g., by fluorescent or dark field microscopic techniques.
  • VIII. Administration and Pharmaceutical compositions
  • Modulators of the polynucleotides or polypeptides of the invention (e.g., antagonists or agonists) can be administered directly to a mammalian subject for modulation of activity of those molecules in vivo. Administration is by any of the routes normally used for introducing a modulator compound into ultimate contact with the tissue to be treated and is well known to those of skill in the art. Although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route.
  • Diseases that can be treated include the following, which include the corresponding reference number from Morrison, DSM-IV Made Easy, 1995: Schizophrenia, Catatonic, Subchronic, (295.21); Schizophrenia, Catatonic, Chronic (295.22); Schizophrenia, Catatonic, Subchronic with Acute Exacerbation (295.23); Schizophrenia, Catatonic, Chronic with Acute Exacerbation (295.24); Schizophrenia, Catatonic, in Remission (295.55); Schizophrenia, Catatonic, Unspecified (295.20); Schizophrenia, Disorganized, Subchronic (295.11); Schizophrenia, Disorganized, Chronic (295.12); Schizophrenia, Disorganized, Subchronic with Acute Exacerbation (295.13); Schizophrenia, Disorganized, Chronic with Acute Exacerbation (295.14); Schizophrenia, Disorganized, in Remission (295.15); Schizophrenia, Disorganized, Unspecified (295.10); Schizophrenia, Paranoid, Subchronic (295.31); Schizophrenia, Paranoid, Chronic (295.32); Schizophrenia, Paranoid, Subchronic with Acute Exacerbation (295.33); Schizophrenia, Paranoid, Chronic with Acute Exacerbation (295.34); Schizophrenia, Paranoid, in Remission (295.35); Schizophrenia, Paranoid, Unspecified (295.30); Schizophrenia, Undifferentiated, Subchronic (295.91); Schizophrenia, Undifferentiated, Chronic (295.92); Schizophrenia, Undifferentiated, Subchronic with Acute Exacerbation (295.93); Schizophrenia, Undifferentiated, Chronic with Acute Exacerbation (295.94); Schizophrenia, Undifferentiated, in Remission (295.95); Schizophrenia, Undifferentiated, Unspecified (295.90); Schizophrenia, Residual, Subchronic (295.61); Schizophrenia, Residual, Chronic (295.62); Schizophrenia, Residual, Subchronic with Acute Exacerbation (295.63); Schizophrenia, Residual, Chronic with Acute Exacerbation (295.94); Schizophrenia, Residual, in Remission (295.65); Schizophrenia, Residual, Unspecified (295.60); Delusional (Paranoid) Disorder (297.10); Brief Reactive Psychosis (298.80); Schizophreniform Disorder (295.40); Schizoaffective Disorder (295.70); Induced Psychotic Disorder (297.30); Psychotic Disorder NOS (Atypical Psychosis) (298.90); Personality Disorders, Paranoid (301.00); Personality Disorders, Schizoid (301.20); Personality Disorders, Schizotypal (301.22); Personality Disorders, Antisocial (301.70); Personality Disorders, Borderline (301.83) and bipolar disorders, maniac, hypomaniac, dysthymic or cyclothymic disorders, substance-induced mood disorders, major depression, psychosis, including paranoid psychosis, catatonic psychosis, delusional psychosis, having schizoaffective disorder, and substance-induced psychotic disorder.
  • In some embodiments, modulators of polynucleotides or polypeptides of the invention can be combined with other drugs useful for treating mental disorders including useful for treating mood disorders, e.g., schizophrenia, bipolar disorders, or major depression. In some preferred embodiments, pharmaceutical compositions of the invention comprise a modulator of a polypeptide of polynucleotide of the invention combined with at least one of the compounds useful for treating schizophrenia, bipolar disorder, or major depression, e.g., such as those described in U.S. Patent Nos. 6,297,262 ; 6,284,760 ; 6,284,771 ; 6,232,326 ; 6,187,752 ; 6,117,890 ; 6,239,162 or 6,166,008 .
  • The pharmaceutical compositions of the invention may comprise a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions of the present invention (see, e.g., Remington's Pharmaceutical Sciences, 17th ed. 1985)).
  • The modulators (e.g., agonists or antagonists) of the expression or activity of the a polypeptide or polynucleotide of the invention, alone or in combination with other suitable components, can be made into aerosol formulations (i.e., they can be "nebulized") to be administered via inhalation or in compositions useful for injection. Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.
  • Formulations suitable for administration include aqueous and non-aqueous solutions, isotonic sterile solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. In the practice of this invention, compositions can be administered, for example, orally, nasally, topically, intravenously, intraperitoneally, or intrathecally. The formulations of compounds can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials. Solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described. The modulators can also be administered as part of a prepared food or drug.
  • The dose administered to a patient, in the context of the present invention should be sufficient to effect a beneficial response in the subject over time. The optimal dose level for any patient will depend on a variety of factors including the efficacy of the specific modulator employed, the age, body weight, physical activity, and diet of the patient, on a possible combination with other drugs, and on the severity of the mental disorder. The size of the dose also will be determined by the existence, nature, and extent of any adverse side effects that accompany the administration of a particular compound or vector in a particular subject.
  • In determining the effective amount of the modulator to be administered a physician may evaluate circulating plasma levels of the modulator, modulator toxicity, and the production of anti-modulator antibodies. In general, the dose equivalent of a modulator is from about 1 ng/kg to 10 mg/kg for a typical subject.
  • For administration, modulators of the present invention can be administered at a rate determined by the LD-50 of the modulator, and the side effects of the modulator at various concentrations, as applied to the mass and overall health of the subject. Administration can be accomplished via single or divided doses.
  • IX. Gene Therapy Applications
  • A variety of human diseases can be treated by therapeutic approaches that involve stably introducing a gene into a human cell such that the gene is transcribed and the gene product is produced in the cell. Diseases amenable to treatment by this approach include inherited diseases, including those in which the defect is in a single or multiple genes. Gene therapy is also useful for treatment of acquired diseases and other conditions. For discussions on the application of gene therapy towards the treatment of genetic as well as acquired diseases, see, Miller, Nature 357:455-460 (1992); and Mulligan, Science 260:926-932 (1993).
  • In the context of the present invention, gene therapy can be used for treating a variety of disorders and/or diseases in which the polynucleotides and polypeptides of the invention has been implicated. For example, compounds, including polynucleotides, can be identified by the methods of the present invention as effective in treating a mental disorder. Introduction by gene therapy of these polynucleotides can then be used to treat, e.g., mental disorders including mood disorders and psychotic disorders.
  • A. Vectors for Gene Delivery
  • For delivery to a cell or organism, the polynucleotides of the invention can be incorporated into a vector. Examples of vectors used for such purposes include expression plasmids capable of directing the expression of the nucleic acids in the target cell. In other instances, the vector is a viral vector system wherein the nucleic acids are incorporated into a viral genome that is capable of transfecting the target cell. In a preferred embodiment, the polynucleotides can be operably linked to expression and control sequences that can direct expression of the gene in the desired target host cells. Thus, one can achieve expression of the nucleic acid under appropriate conditions in the target cell.
  • B. Gene Delivery Systems
  • Viral vector systems useful in the expression of the nucleic acids include, for example, naturally occurring or recombinant viral vector systems. Depending upon the particular application, suitable viral vectors include replication competent, replication deficient, and conditionally replicating viral vectors. For example, viral vectors can be derived from the genome of human or bovine adenoviruses, vaccinia virus, herpes virus, adeno-associated virus, minute virus of mice (MVM), HIV, sindbis virus, and retroviruses (including but not limited to Rous sarcoma virus), and MoMLV. Typically, the genes of interest are inserted into such vectors to allow packaging of the gene construct, typically with accompanying viral DNA, followed by infection of a sensitive host cell and expression of the gene of interest.
  • As used herein, "gene delivery system" refers to any means for the delivery of a nucleic acid of the invention to a target cell. In some embodiments of the invention, nucleic acids are conjugated to a cell receptor ligand for facilitated uptake (e.g., invagination of coated pits and internalization of the endosome) through an appropriate linking moiety, such as a DNA linking moiety (Wu et al., J. Biol. Chem. 263:14621-14624 (1988); WO 92/06180 ). For example, nucleic acids can be linked through a polylysine moiety to asialo-oromucocid, which is a ligand for the asialoglycoprotein receptor of hepatocytes.
  • Similarly, viral envelopes used for packaging gene constructs that include the nucleic acids of the invention can be modified by the addition of receptor ligands or antibodies specific for a receptor to permit receptor-mediated endocytosis into specific cells (see, e.g., WO 93/20221 , WO 93/14188 , and WO 94/06923 ). In some.embodiments of the invention, the DNA constructs of the invention are linked to viral proteins, such as adenovirus particles, to facilitate endocytosis (Curiel et al., Proc. Natl. Acad. Sci. U.S.A. 88:8850-8854 (1991)). In other embodiments, molecular conjugates of the instant invention can include microtubule inhibitors ( WO/9406922 ), synthetic peptides mimicking influenza virus hemagglutinin (Plank et al., J. Biol. Chem. 269:12918-12924 (1994)), and nuclear localization signals such as SV40 T antigen ( WO93/19768 ).
  • Retroviral vectors are also useful for introducing the nucleic acids of the invention into target cells or organisms. Retroviral vectors are produced by genetically manipulating retroviruses. The viral genome of retroviruses is RNA. Upon infection, this genomic RNA is reverse transcribed into a DNA copy which is integrated into the chromosomal DNA of transduced cells with a high degree of stability and efficiency. The integrated DNA copy is referred to as a provirus and is inherited by daughter cells as is any other gene. The wild type retroviral genome and the proviral DNA have three genes: the gag, the pol and the env genes, which are flanked by two long terminal repeat (LTR) sequences. The gag gene encodes the internal structural (nucleocapsid) proteins; the pol gene encodes the RNA directed DNA polymerase (reverse transcriptase); and the env gene encodes viral envelope glycoproteins. The 5' and 3' LTRs serve to promote transcription and polyadenylation of virion RNAs. Adjacent to the 5' LTR are sequences necessary for reverse transcription of the genome (the tRNA primer binding site) and for efficient encapsulation of viral RNA into particles (the Psi site) (see, Mulligan, In: Experimental Manipulation of Gene Expression, Inouye (ed), 155-173 (1983); Mann et al., Cell 33:153-159 (1983); Cone and Mulligan, Proceedings of the National Academy of Sciences, U.S.A., 81:6349-6353 (1984)).
  • The design of retroviral vectors is well known to those of ordinary skill in the art. In brief, if the sequences necessary for encapsidation (or packaging of retroviral RNA into infectious virions) are missing from the viral genome, the result is a cis-acting defect which prevents encapsidation of genomic RNA. However, the resulting mutant is still capable of directing the synthesis of all virion proteins. Retroviral genomes from which these sequences have been deleted, as well as cell lines containing the mutant genome stably integrated into the chromosome are well known in the art and are used to construct retroviral vectors. Preparation of retroviral vectors and their uses are described in many publications including, e.g., European Patent Application EPA 0 178 220 ; U.S. Patent 4,405,712 , Gilboa Biotechniques 4:504-512 (1986); Mann et al., Cell 33:153-159 (1983); Cone and Mulligan Proc. Natl. Acad. Sci. USA 81:6349-6353 (1984); Eglitis et al. Biotechniques 6:608-614 (1988); Miller et al. Biotechniques 7:981-990 (1989); Miller (1992) supra; Mulligan (1993), supra; and WO 92/07943 .
  • The retroviral vector particles are prepared by recombinantly inserting the desired nucleotide sequence into a retrovirus vector and packaging the vector with retroviral capsid proteins by use of a packaging cell line. The resultant retroviral vector particle is incapable of replication in the host cell but is capable of integrating into the host cell genome as a proviral sequence containing the desired nucleotide sequence. As a result, the patient is capable of producing, for example, a polypeptide or polynucleotide of the invention and thus restore the cells to a normal phenotype.
  • Packaging cell lines that are used to prepare the retroviral vector particles are typically recombinant mammalian tissue culture cell lines that produce the necessary viral structural proteins required for packaging, but which are incapable of producing infectious virions. The defective retroviral vectors that are used, on the other hand, lack these structural genes but encode the remaining proteins necessary for packaging. To prepare a packaging cell line, one can construct an infectious clone of a desired retrovirus in which the packaging site has been deleted. Cells comprising this construct will express all structural viral proteins, but the introduced DNA will be incapable of being packaged. Alternatively, packaging cell lines can be produced by transforming a cell line with one or more expression plasmids encoding the appropriate core and envelope proteins. In these cells, the gag, pol, and env genes can be derived from the same or different retroviruses.
  • A number of packaging cell lines suitable for the present invention are also available in the prior art. Examples of these cell lines include Crip, GPE86, PA317 and PG13 (see Miller et al., J. Virol. 65:2220-2224 (1991)). Examples of other packaging cell lines are described in Cone and Mulligan Proceedings of the National Academy of Sciences, USA, 81:6349-6353 (1984); Danos and Mulligan Proceedings of the National Academy of Sciences, USA, 85:6460-6464 (1988); Eglitis et al. (1988), supra; and Miller (1990), supra.
  • Packaging cell lines capable of producing retroviral vector particles with chimeric envelope proteins may be used. Alternatively, amphotropic or xenotropic envelope proteins, such as those produced by PA317 and GPX packaging cell lines may be used to package the retroviral vectors.
  • In some embodiments of the invention, an antisense polynucleotide is administered which hybridizes to a gene encoding a polypeptide of the invention. The antisense polypeptide can be provided as an antisense oligonucleotide (see, e.g., Murayama et al., Antisense Nucleic Acid Drug Dev. 7:109-114 (1997)). Genes encoding an antisense nucleic acid can also be provided; such genes can be introduced into cells by methods known to those of skill in the art. For example, one can introduce an antisense nucleotide sequence in a viral vector, such as, for example, in hepatitis B virus (see, e.g., Ji et al., J. Viral Hepat. 4:167-173 (1997)), in adeno-associated virus (see, e.g., Xiao et al., Brain Res. 756:76-83 (1997)), or in other systems including, but not limited, to an HVJ (Sendai virus)-liposome gene delivery system (see, e.g., Kaneda et al., Ann. NY Acad. Sci. 811:299-308 (1997)), a "peptide vector" (see, e.g., Vidal et al., CR Acad. Sci III 32:279-287 (1997)), as a gene in an episomal or plasmid vector (see, e.g., Cooper et al., Proc. Natl. Acad. Sci. U.S.A. 94:6450-6455 (1997), Yew et al. Hum Gene Ther. 8:575-584 (1997)), as a gene in a peptide-DNA aggregate (see, e.g., Niidome et al., J. Biol. Chem. 272:15307-15312 (1997)), as "naked DNA" (see, e.g., U.S. patent Nos. 5,580,859 and 5,589,466 ), in lipidic vector systems (see, e.g., Lee et al., Crit Rev Ther Drug Carrier Syst. 14:173-206 (1997)), polymer coated liposomes ( U.S. patent Nos. 5,213,804 and 5,013,556 ), cationic liposomes ( Epand et al., U.S. patent Nos. 5,283,185 ; 5,578,475 ; 5,279,833 ; and 5,334,761 ), gas filled microspheres ( U.S. patent No. 5,542,935 ), ligand-targeted encapsulated macromolecules ( U.S. patent Nos. 5,108,921 ; 5,521,291 ; 5,554,386 ; and 5,166,320 ).
  • Upregulated transcripts listed in the biomarker tables herein which are correlated with mental disorders may be targeted with one or more short interfering RNA (siRNA) sequences that hybridize to specific sequences in the target, as described above. Targeting of certain brain transcripts with siRNA in vivo has been reported, for example, by Zhang et al., J. Gene. Med., 12:1039-45 (2003), who utilized monoclonal antibodies against the transferrin receptor to facilitate passage of liposome-encapsulated siRNA molecules through the blood brain barrier. Targeted siRNAs represent useful therapeutic compounds for attenuating the over-expressed transcripts that are associated with disease states, e.g., MDD, BP, and other mental disorders.
  • In another embodiment, conditional expression systems, such as those typified by the tet-regulated systems and the RU-486 system, can be used (see, e.g., Gossen & Bujard, PNAS 89:5547 (1992); Oligino et al., Gene Ther. 5:491-496 (1998); Wang et al., Gene Ther. 4:432-441 (1997); Neering et al., Blood 88:1147-1155 (1996); and Rendahl et al., Nat. Biotechnol. 16:757-761 (1998)). These systems impart small molecule control on the expression of the target gene(s) of interest.
  • In another embodiment, stem cells engineered to express a transcript of interest can implanted into the brain.
  • C. Pharmaceutical Formulations
  • When used for pharmaceutical purposes, the vectors used for gene therapy are formulated in a suitable buffer, which can be any pharmaceutically acceptable buffer, such as phosphate buffered saline or sodium phosphate/sodium sulfate, Tris buffer, glycine buffer, sterile water, and other buffers known to the ordinarily skilled artisan such as those described by Good et al. Biochemistry 5:467 (1966).
  • The compositions can additionally include a stabilizer, enhancer, or other pharmaceutically acceptable carriers or vehicles. A pharmaceutically acceptable carrier can contain a physiologically acceptable compound that acts, for example, to stabilize the nucleic acids of the invention and any associated vector. A physiologically acceptable compound can include, for example, carbohydrates, such as glucose, sucrose or dextrans; antioxidants, such as ascorbic acid or glutathione; chelating agents; low molecular weight proteins or other stabilizers or excipients. Other physiologically acceptable compounds include wetting agents, emulsifying agents, dispersing agents, or preservatives, which are particularly useful for preventing the growth or action of microorganisms. Various preservatives are well known and include, for example, phenol and ascorbic acid. Examples of carriers, stabilizers, or adjuvants can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, PA, 17th ed. (1985).
  • D. Administration of Formulations
  • The formulations of the invention can be delivered to any tissue or organ using any delivery method known to the ordinarily skilled artisan. In some embodiments of the invention, the nucleic acids of the invention are formulated in mucosal, topical, and/or buccal formulations, particularly mucoadhesive gel and topical gel formulations. Exemplary permeation enhancing compositions, polymer matrices, and mucoadhesive gel preparations for transdermal delivery are disclosed in U.S. Patent No. 5,346,701 .
  • E. Methods of Treatment
  • The gene therapy formulations of the invention are typically administered to a cell. The cell can be provided as part of a tissue, such as an epithelial membrane, or as an isolated cell, such as in tissue culture. The cell can be provided in vivo, ex vivo, or in vitro.
  • The formulations can be introduced into the tissue of interest in vivo or ex vivo by a variety of methods. In some embodiments of the invention, the nucleic acids of the invention are introduced into cells by such methods as microinjection, calcium phosphate precipitation, liposome fusion, or biolistics. In further embodiments, the nucleic acids are taken up directly by the tissue of interest.
  • In some embodiments of the invention, the nucleic acids of the invention are administered ex vivo to cells or tissues explanted from a patient, then returned to the patient. Examples of ex vivo administration of therapeutic gene constructs include Nolta et al., Proc Natl. Acad. Sci. USA 93(6):2414-9 (1996); Koc et al., Seminars in Oncology 23 (1):46-65 (1996); Raper et al., Annals of Surgery 223(2):116-26 (1996); Dalesandro et al., J. Thorac. Cardi. Surg., 11(2):416-22 (1996); and Makarov et al., Proc. Natl. Acad. Sci. USA 93(1):402-6 (1996).
  • X. Diagnosis of mood disorders and psychotic disorders
  • The present invention also provides methods of diagnosing mood disorders (such as major depression or bipolar disorder), psychotic disorders (such as schizophrenia), or a predisposition of at least some of the pathologies of such disorders. Diagnosis involves determining the level of a polypeptide or polynucleotide of the invention in a patient and then comparing the level to a baseline or range. Typically, the baseline value is representative of a polypeptide or polynucleotide of the invention in a healthy person not suffering from a mood disorder or a psychotic disorder or under the effects of medication or other drugs. Variation of levels of a polypeptide or polynucleotide of the invention from the baseline range (either up or down) indicates that the patient has a mood disorder or a psychotic disorder or at risk of developing at least some aspects of a mood disorder or a psychotic disorder. In some embodiments, the level of a polypeptide or polynucleotide of the invention are measured by taking a blood, urine or tissue sample from a patient and measuring the amount of a polypeptide or polynucleotide of the invention in the sample using any number of detection methods, such as those discussed herein.
  • Antibodies can be used in assays to detect differential protein expression in patient samples, e.g., ELISA assays, immunoprecipitation assays, and immunohistochemical assays. PCR assays can be used to detect expression levels of nucleic acids, as well as to discriminate between variants in genomic structure, such as insertion/deletion mutations (e.g., PSPHL).
  • In the case where absence of gene expression is associated with a disorder, the genomic structure of a gene such as PSPHL can be evaluated with known methods such as PCR to detect deletion or insertion mutations associated with disease suspectibility. Conversely, the presence of mRNA or protein corresponding to the PSPHL gene would indicate that an individual does not have the PSPHL deletion associated with susceptibility to BP. Thus, diagnosis can be made by detecting the presence or absence of mRNA or protein, or by examining the genomic structure of the gene. Any combination of exons or non-transcribed regions can be used to detect the deletion allele. For example, the presence of exon 4 but not exons 1, 2, and/or 3 would indicate the presence of the deletion allele. Similarly, deletion of the promoter region would indicate the deletion allele. Any significant mRNA detection, especially detection of an mRNA comprising exons 1, 2, and/or 3, would indicate the absence of the deletion allele, which is not transcribed due to the promoter deletion.
  • Single nucleotide polymorphism (SNP) analysis is also useful for detecting differences between alleles of the polynucleotides (e.g., genes) of the invention. SNPs linked to genes encoding polypeptides of the invention are useful, for instance, for diagnosis of diseases (e.g., mood disorders such as bipolar disease, major depression, and schizophrenia disorders) whose occurrence is linked to the gene sequences of the invention. For example, if an individual carries at least one SNP linked to a disease-associated allele of the gene sequences of the invention, the individual is likely predisposed for one or more of those diseases. If the individual is homozygous for a disease-linked SNP, the individual is particularly predisposed for occurrence of that disease. In some embodiments, the SNP associated with the gene sequences of the invention is located within 300,000; 200,000; 100,000; 75,000; 50,000; or 10,000 base pairs from the gene sequence.
  • Various real-time PCR methods can be used to detect SNPs, including, e.g., Taqman or molecular beacon-based assays (e.g., U.S. Patent Nos. 5,210,015 ; 5,487,972 ; Tyagi et al., Nature Biotechnology 14:303 (1996); and PCT WO 95/13399 are useful to monitor for the presence of absence of a SNP. Additional SNP detection methods include, e.g., DNA sequencing, sequencing by hybridization, dot blotting, oligonucleotide array (DNA Chip) hybridization analysis, or are described in, e.g., U.S. Patent No. 6,177,249 ; Landegren et al., Genome Research, 8:769-776 (1998); Botstein et al., Am J Human Genetics 32:314-331 (1980); Meyers et al., Methods in Enzymology 155:501-527 (1987); Keen et al., Trends in Genetics 7:5 (1991); Myers et al., Science 230:1242-1246 (1985); and Kwok et al., Genomics 23:138-144 (1994). PCR methods can also be used to detect deletion/insertion polymorphisms, such as the deletion polymorphism of the PSPHL gene associated with suspectibility to BP.
  • In some embodiments, the level of the enzymatic product of a polypeptide or polynucleotide of the invention is measured and compared to a baseline value of a healthy person or persons. Modulated levels of the product compared to the baseline indicates that the patient has a mood disorder or a psychotic disorder or is at risk of developing at least some aspects of a mood disorder or a psychotic disorder. Patient samples, for example, can be blood, urine or tissue samples.
  • It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.
  • EXAMPLES Example 1: Identification of FGF pathway genes dysregulated in MDD
  • Major depressive disorder (MDD) and bipolar disorder (BP) are affective diseases that strike a significant proportion of the population. These complex genetic disorders arise from the interplay of vulnerability genes and environmental stressors, impacting neural circuits that control mood. Beyond the role of limbic structures, mood disorders are hypothesized to involve aberrant activity of the cerebral cortex. Thus, imaging techniques have implicated the dorsolateral prefrontal (DLPFC) and anterior cingulate (AnCg) cortices in mood disorders since affected subjects display changes in volumetric measurements (Harrison, P. J. Brain 125, 1428-49 (2002).) and altered activity in response to a cognitive challenge. (Kruger, S., Seminowicz, D., Goldapple, K., Kennedy, S. H. & Mayberg, H. S. Biol Psychiatry 54, 1274-83 (2003)) Using a candidate approach, studies have demonstrated altered cortical expression of specific neurotransmitter- and stress-related genes in affective illness. However, the full extent of the alteration in cortical activity had not been described, nor had an unbiased "discovery" approach been applied to characterize it.
  • We have applied microarray technology to the study of DLPFC and AnCg in post-mortem samples from MDD, BP and non-psychiatric controls. This represents the first transcriptional profiling study demonstrating significant alterations of gene expression in major depression, and the first one contrasting the two major mood disorders (MDD and BP) with the same group of controls. Here we report the dysregulation of fibroblast growth factor (FGF) system transcripts specifically in MDD.
  • Human Studies
  • The studies were carried out in a carefully selected cohort of postmortem human brains, and replicated in a separate cohort (see Table 1a and 1b). The Affymetrix HG-133A array contains probe sets for 21 FGF system transcripts, including all 4 receptors (FGFR1, 2, 3, 4) and 12 FGF peptide ligands (FGF1, 2, 3, 5, 6, 7, 8, 9, 12, 13, 14, 17, 18, 20, 21, 22, 23). Of these only 10 were reliably detected in the regions assayed and include three of the FGF receptors (FGFR1, 2, and 3) and seven FGF ligands (FGF1, 2, 7, 9, 12, 13,14). Of the ten FGF transcripts reliably detected, seven were significantly altered in one of the two regions studied- four were significantly differentially expressed in the DLPFC of MDD subjects, including 2 FGF receptors (FGFR2 and 3) and 2 FGF ligands (FGF1 and 9); and six were significantly differentially expressed in the AnCg including two receptors (FGFR2 and 3) and four ligands (FGF1, 2, 9 and 12). The probability that this family of molecules would have emerged by chance, based on a hypergeometric distribution, is p<0.001. These data are summarized in Table 2, which also reports on the transcripts confirmed by real-time PCR analysis and/or those replicated in a second independent cohort of MDD and control subjects. Importantly, none of the above transcripts were observed to be differentially expressed in BP by microarray in either cohort or by real-time PCR analysis, demonstrating the specificity of this dysregulation for MDD.
  • Given this selective dysregulation of FGF system transcripts in MDD we asked whether these changes might be secondary to antidepressant therapy since a subset of the subjects was on antidepressants, and the majority of those were on specific serotonin reuptake inhibitors (SSRI's). Thus, we separated our microarray data into MDD subjects prescribed SSRI's (n=5) and those not prescribed SSRI's (n=4) for statistical comparisons. This analysis showed that FGFR3 and FGF2 had a tendency toward up-regulation (p=0.15 and 0.07, respectively) and that FGF 9 showed a tendency toward down-regulation (p=0.12) by SSRI treatment, all opposing the directionality of dysregulation we see in MDD subjects relative to controls. No other FGF system transcripts approached significant differences in this analysis. These data strongly suggest that our observations are not secondary to SSRI treatment. Furthermore, the observed attenuation of FGF transcript dysregulation in the SSRI prescribed group suggests that the normalization of FGF system might be one mechanism of action of this class of drugs since several FGF transcripts appear to be altered in severe depression and are partially reversed by SSRI therapy.
  • Rodent Anatomical Studies; Effect of Fluoxetine.
  • We studied the anatomical expression of FGFR2 in rats subjected to chronic fluoxetine treatment relative to controls (Fig. 1). FGFR2 mRNA expression was quantified in the retrosplenial cortex and in the hippocampus since this area has recently been implicated in the mode of action of antidepressants (Santarelli, L. et al. )). Results show that FGFR2 message is significantly increased by fluoxetine in all hippocampal subfields (CA1, CA2, CA3, and dentate gyrus) and the retrosplenial cortex (Fisher's PLSD, p=0.0049).
  • The implication of the FGF system in MDD has emerged from our studies utilizing microarray technology to study human psychiatric illness. Other growth factors have been hypothesized to contribute to the etiology and maintenance of such illnesses and can offer a framework in which to place our own findings. Most notably, brain derived neurotrophic factor (BDNF) has been repeatedly implicated in MDD, BP and in SZ. BDNF mRNA levels are reportedly decreased in the DLPFC (Weickert, C. S., et al., )) of schizophrenics and BDNF protein levels are decreased in serum of MDD (Shimizu, E. et al. Biol Psychiatry 54, 70-5 (2003)) patients. Furthermore, BDNF expression is regulated by antipsychotic (Chlan-Fourney, J. et al., Brain Res 954, 11-20 (2002)) and antidepressant drugs (Shimizu, E. et al. Biol Psychiatry 54, 70-5 (2003)), (Dias, B. G., et al., )). A smaller volume of literature implicates other growth factors, including nerve growth factor (Parikh, V., Evans, D. R., Khan, M. M. & Mahadik, S. P. )), epidermal growth factor (Futamura, T. et al. )) and neurotrophin-3 (Hock, C. et al. )) in psychiatric illness.
  • Growth factors play significant roles in development and maintenance of the central nervous system. In the developing brain, they are involved in specific neuronal terminal differentiation and migration to appropriate subfields. In the adult brain they are critical in neuronal survival, axonal branching and synaptic plasticity. Specifically, FGF2 (Viti, J., Gulacsi, A. & Lillien, L. J Neurosci 23, 5919-27 (2003)) and FGF8 (Gunhaga, L. et al. )) have been shown to interact with Wnt in the development of the cortex in mouse and chick embryos, respectively. In the adult brain, FGF2 promotes neuronal survival and axonal branching (Abe, K. & Saito, H. Pharmacol Res 43, 307-12 (2001)) and its expression is modulated by stress (Molteni, R. et al., )).
  • Together, these results lead to the novel hypothesis that dysregulation of the FGF system contributes to either the vulnerability to MDD or the expression of the illness, and that antidepressants might attenuate this dysregulation. Animal models will be crucial for defining the involvement of the FGF system in emotionality and elucidating its role in neural plasticity and antidepressant action.
  • Example 2: Identification of novel insertion/deletion polymorphism in PSPHL gene and association of deletion mutation with BP susceptibility
  • Evaluation of PSPHL expression in anterior cingulated cortex by quantitative RT-PCR reveals that PSPHL shows a dichotomous (present or absent) pattern of expression among individuals. In our first cohort, none of the 9 BPD patients (0%) shows PSPHL expression, while 7 out of 11 MDD patients (64%) and 8 out of 20 controls (40%) show sufficient expression of PSPHL. The probability of distribution of the present/absent expression pattern between BPD and controls is 0.018, and between MDD and controls is 0.105 based on the Fisher exact test. To assure this significant contrast between BPD and controls, we measured PSPHL expression in an additional 9 BPD and 20 control individuals (for a total of 18 BPD and 40 controls) using quantitative RT-PCR. In this larger sample set, none of the 18 BPD patients (0%) shows any expression of PSPHL, while 16 out of 40 controls (40%) show sufficient expression of the transcript (p value = 0.0008).
  • The fact that PSPHL shows dichotomous present/absent pattern of expression among individuals with brain-wide consistency suggests genetic variation in its regulation. Since genomic organization of PSPHL has not been characterized (Planitzer, supra (1998)), we have identified genomic organization of PSPHL as shown in Figure 14. The PSPHL gene consists of 4 exons. Exons 1, 2, 3 and 4 are 213 bp, 114 bp, 122 bp and 501 bp, in length, respectively, and span introns 1, 2 and 3 (3221bp, 829 bp and 11939 bp, in length, respectively). Further, the PSPHL gene has two alternative transcripts, one of which utilizes the exons 1-4 (PSPHL-A in figure 14), while another utilizes the exons 1, 2 and 4 (PSPHL-B). We have also identified an insertion/deletion polymorphism at the PSPHL locus. The deleted genomic region spans more than 30 kb, including the promoter region and the exons 1, 2 and 3 of PSPHL gene. This genetic variance explains the present/absent pattern of the PSPHL expression. An over-representation of the deletion allele resulting in the absence of PSPHL expression increases susceptibility to BPD.
  • PSPHL and PSPH are highly homologous, but appear to be different genes, which are about 200kb apart from each other on chromosome 7p11.2 region. Especially, exons 2-4 of PSPHL are highly homologous to exons 4 and 8 of PSPH gene. Predicted amino acid sequences of PSPH, PSPHL-A and PSPHL-B are shown in Figure 15. PSPHL-A and PSPHL-B share N-terminal 57 common amino acids, transcribed from exons 1 and 2. PSPHL-A has unique C-terminal 36 amino acids, transcribed from exon3, while PSPHL-B has unique C-terminal 17 amino acids, transcribed from exon 4. PSPH and PSPHL-A&B have 31 amino acids in common. The common amino acids locates at the N-terminal end of PSPH and middle region (25th - 56th amino acids) of PSPHL-A and B. The common region contains consensus phosphorylation site of Na/K ATPase and casein kinase II phospholyration site. Based on the similarity in the structure, PSPHL shares some function with PSPH gene.
  • PSPH is the rate limiting enzyme for serine synthesis. PSPH has haloacid dehalogenase-like hydrolase domain, which is responsible for the activity. Greater than 90% of L-serine in brain is formed via the phosphorylated pathway. PSPH may be dimeric from of the enzyme with a monomeric molecular weight of 26kDa. L-serine is converted to sphingomyelins and gangliosides, as well as L-glycine and D-serine, both of which act as coagonist for NMDA receptor associated glycine binding site. L-glycine is also an agonist for strychnine-sensitive glycine receptor (Figure 16). PSPHL is involved in serine amino acid metabolic pathway, and may involved in other pathways as well.
  • Example 3: Post-natal injection of FGF2
  • This Example shows that neonatal administration of FGF-2 affects long-term alterations in hippocampal volume, emotional reactivity and learning and memory. Sprague-Dawley rats were injected with either vehicle or FGF-2 (20 ng/g, s.c.) on postnatal day 2 (PD2). Three weeks after injection we evaluated dentate gyrus volume and cell counts by Nissl staining. We also assessed neurogenesis by BrdU and Ki-67 immunohistochemistry at the 23 day time point. In adult rats, we tested locomotor activity, anxiety behavior and learning and memory. The animals were sacrificed, and the brains collected for in situ hybridization (FGF markers, stress markers). Results to date have shown the following: FGF-2 injected rats exhibited a 10.5% increase in dentate gyrus volume. The results show that FGF-2 significantly increased locomotor activity over controls in a novel environment. Increased activity in response to novelty has been associated with a host of other measures including decreased anxiety-like behavior. Furthermore, adult rats that received FGF-2 as neonates also performed significantly better than controls in the Morris water maze.
  • Example 4: FGF2 expression
  • While evidence has linked growth factors such as BDNF to environmental complexity (EC), responsiveness to stress, and antidepressant action, few studies focused on the role of the FGF system in emotional reactivity. Recent data from our laboratory suggest that a single postnatal injection of FGF-2 significantly alters locomotor activity in response to a novel environment (Turner et al., SFN abstracts 2004). Since increased responsiveness to novelty is associated with decreased anxiety-like behavior, we propose that FGF-2 may be correlated with other indices of emotionality. We tested the hypothesis that changes in emotionality associated with EC may be related to FGF-2 gene expression in the hippocampus. Young adult male Sprague-Dawley rats were either exposed to a complex environment for 21 days or to standard cages. Following this treatment, rats were returned to standard cages for two weeks. Brains were then processed for neurogenesis by BrdU and Ki-67 immunohistochemistry. Another group of rats was tested in the elevated plus-maze (EPM) and then sacrificed for in situ hybridization. Compared to controls, EC rats showed significantly less anxiety-like behavior in the EPM and exhibited a 23% increase in FGF-2 expression in the hippocampus. There was a significant positive correlation between FGF-2 mRNA levels in hippocampal CA2 and time spent in the open arms of the EPM. Whether these results relate to levels of neurogenesis in the hippocampus is currently being determined. These findings are consistent with our observations in human postmortem brains (Evans et al, SfN Abstracts 2004) showing that expression of several members of the FGF family is decreased in major depression. Together, these findings implicate the FGF system in emotionality and mood disorders.
  • Example 5. CYCLIC AMP SIGNALING PATHWAY GENES DIFFERENTIALLY EXPRESSED IN BPD AND/OR MDD PATIENTS
  • Two independent cohorts A and B were analyzed separately in this study. Cohort A consisted of 22 subjects including 7 healthy control subjects, 6 patients with BPD and 9 patients with MDD. Cohort B consisted of 12 subjects including 5 MDD and 7 controls. All subjects in this study did not have specific agonal conditions including hypoxia, coma, pyrexia, seizure, dehydration, hypoglycemia, multi-organ failure, skull fracture, ingestion of neurotoxic substances or prolonged agonal duration, which is known to affect tissue pH, RNA integrity and gene expression profile in postmortem brain, and showed brain tissue pH of more than 6.5. In order to detect reliable gene expression differences between diagnostic groups, we performed experimentally as well as biologically replicated experiments as follows. Experiment 1: Total RNA was extracted from AnCg, DLPFC and CB of the Cohort A, and purified with silica-based mini-spin columns (Qiagen RNeasy Mini Kit, Valencia, California). The oligonucleotide microarray experiments were carried out following the manufacturer's protocol (Affymetrix, Santa Clara, CA). For technical replication, each of RNA samples was run on Affymetrix U95Av2 GeneChips at two laboratories. Experiment 2: For further technical replication, samples from the 22 subjects from cohort A were reanalyzed in AnCg and DLPFC utilizing U133A GeneChips at two laboratories. Experiment 3: Samples from the additional cohort B were analyzed on U133A GeneChips in AnCg and DLPFC at two laboratories. Signal intensity data was extracted with Robust Multi-array Average (RMA) for each probe set and each subject. Gene-wise Pearson's correlation coefficients between experimental duplicates were calculated, and only the genes significantly correlated between experimental duplicates were considered to be reliably detectable genes, and subjected to the downstream analyses. For these reliably detectable genes, mixed-model multivariate ANOVA analyses were employed utilizing Partek Pro 6.0 (Partek, St.Charles, MO) to adjust the effect of the diagnostic classification (BPD, MDD, control) for possible confounders, including site for experiment, experimental batch, and gender. Post hoc tests (least-squares difference) were run to generate p value for the differences between case and control means, and false discovery rate multiple comparison corrections at the level of accepting 5% false positives was applied to each ANOVA result. When the p value passed false discovery rate multiple testing correction at the level of accepting 5% false positives and percent fold change exceeds 20%, the genes were consider to be significantly differentially expressed between case and control groups. Also, in order to evaluate more subtle but consistent expression differences between case and control groups, we also selected genes passed p value of 0.05 regardless of FDR correction and %FC >10% on both experimental duplicates utilizing U95Av2 or U133A GeneChips.
  • Figure 26A and Table 14 summarize cAMP signaling pathway related genes which were differentially expressed in anterior cingulate cortex (AnCg) of bipolar disorder (BPD) patients compared with controls. Among GPCRs coupled with G protein inhibitory subunit (Gi), which inhibits adenylate cyclase activity, neuropeptide Y receptor 1 (NPY1) was significantly increased in AnCg of BPD. The ligand, neuropeptide Y was also significantly increased in AnCg of BPD. Gi-linked metabotropic glutamate receptor 3 (GRM3) was also increased in AnCg as well as dorsolateral prefrontal cortex (DLPFC) of BPD. Somatostatin (SST), a ligand for Gi-coupled GPCR, was significantly increased in AnCg in our microarray data.
  • Contrasted to the finding in AnCg, SST mRNA expression was decreased in DLPFC of BPD as well as major depressive disorder (MDD). Adrenergic beta-1 receptor (ADRB1) was decreased by 13-18% in DLPFC of BPD, although the change did not reach the significant criteria (Table 17). Proenkephalin (PENK), a ligand for Gi-coupled GPCR, was not altered in AnCg and DLPFC of BPD and MDD, but significantly increased in CB of both BPD and MDD.
  • Messenger RNA expression level of G protein alpha subunit inhibitory peptide 1 (GNAI1) and phosphodiesterase 1A (PDE1A) were significantly increased in AnCg of BPD patients. Protein kinase A inhibitor alpha (PKIA) and cyclin dependent kinase 5 (CDK5), phosphodiesterase 8A (PDE8A) and protein phosphatase 1, catalytic subunit, alpha (PPP1CA) did not reach significant criteria, but were increased by 10%-20% in AnCg of BPD. Thus, mRNA expression of molecules suppressing cAMP concentration and PKA activity were generally increased in BPD, while molecules activating cAMP signaling (Gs-coupled GPCR, Gs, adenylate cyclase, protein kinase A) did not show significant alteration at the transcript level.
  • Figure 26 and Table 14 summarize cAMP signaling pathway related genes which were differentially expressed in AnCg of MDD patients compared with controls. Gi-linked endothelial differentiation GPCR 1 (EDG1) was significantly decreased in AnCg of MDD. Regulator of G protein signaling 20 (RGS20), phosphodiesterase 8A (PDE8A), and protein phosphatase 1 regulatory subunit 3C (PPP1R3C) showed significantly lower expression in AnCg of MDD patients compared with controls. Expression levels of PDE8A and PPP1R3C mRNAs were significantly lower also in DLPFC of MDD (Table 15). Significant decrease in RGS20 expression in MDD was observed also in CB (Table 16). Thus, mRNA expression of molecules suppressing cAMP concentration and PKA activity were generally decreased in MDD, while the molecules activating cAMP signaling did not show significant alteration at the transcript level.
  • PHOSPHATIDYLINOSITOL SIGNALING PATHWAY
  • Figure 26C and Table 14 summarize phosphatidylinositol signaling (PI) pathway related genes which were differentially expressed in AnCg of BPD patients compared with controls. Gq-linked tachykinin (neuropeptide K) receptor 2 (TACR2) was significantly decreased in AnCg of BPD. Messenger RNA expression of inositol polyphosphate-1-phosphatase (INPP1) was significantly higher, while CDP-diacylglycerol synthase 1 (CDS1), a regulatory subunit of class I phosphatidylinositol 3 kinase (PIK3R1) and protein kinase C iota (PKCI) were significantly lower in AnCg of BPD patients compared with control group. Inositol 1, 4, 5-trisphosphate 3-kinase B (ITPKB) and catalytic beta subunit of class II phosphatidylinositol 3 kinase (PIK3C2B) did not reach significance criteria, but increased by 10%-20% in AnCg of BPD.
  • Figure 26D and Table 14 summarize PI signaling pathway related genes which were differentially expressed in AnCg of MDD patients compared with controls. Gq-linked neurotensin receptor 2 (NTSR2) and endothelin receptor type B (EDNRB) were significantly decreased in AnCg of MDD. Messenger RNA expression of inositol polyphosphate-5-phosphatase F (INPP5F) was significantly higher, while inositol 1, 4, 5-trisphosphate 3-kinase B (ITPKB) and catalytic alpha subunit of class II phosphatidylinositol 3 kinase (PIK3C2A) were significantly lower in AnCg of MDD compared with control. Inositol polyphosphate-5-phosphatase A (INPP5A), protein kinase C beta 1 (PKCB 1) and inositol 1, 4, 5-triphosphate receptor type 1 (ITPR1) did not reach the significance criteria, but increased by 10-20% in AnCg of MDD patients. Significant decrease in ITPKB mRNA expression was observed also in DLPFC of MDD (Table 15). Neither G protein alpha q subunit nor phospholipase C beta mRNAs were altered in any of the brain regions of the disorder groups.
  • OTHER G PROTEIN-COUPLED RECEPTORS
  • Among all GPCRs, the most consistent differential expression patterns throughout our experiments were observed in G protein-coupled receptor family C, group 5, member B (GPRC5B) and G protein-coupled receptor 37 (GPR37). GPRC5B was significantly increased in AnCg and DLPFC of BPD. GPRC5B was significantly decreased in AnCg, DLPFC and CB of MDD patients. A significant decrease of GPRC5B in AnCg and DLPFC of MDD patients was replicated by the experiments utilizing another independent cohort B. GPR37 was also significantly increased in AnCg of BPD, and significantly decreased in AnCg, DLPFC and CB of MDD.
  • QUANTITATIVE RT-PCR
  • For further technical evaluation of the microarray data, we evaluated mRNA expression levels by real-time quantitative reverse transcriptase PCR (qRT-PCR) for the following 7 genes, in anterior cingulate cortex (AnCg): Somatostatin (SST), neuropeptide Y (NPY), G protein-coupled receptor C-5-B (GPRC5B), G protein-coupled receptor 37 (GPR37), regulator of G-protein signaling 20 (RGS20), inositol polyphosphate-1-phosphatase (INPP1) and protein phosphatase 1 regulatory subunit 3C(PPP1R3C).
  • In consistent with microarray data, qRT-PCR data showed that mRNA expressions of neuropeptide Y (NPY), G protein-coupled receptor C-5-B (GPRC5B), G protein-coupled receptor 37 (GPR37), inositol polyphosphate-1-phosphatase (INPP1) were significantly increased in AnCg of BPD, and expression level of GPRCSB, GPR37, regulator of G-protein signaling 20 (RGS20) and protein phosphatase 1 regulatory subunit 3C (PPP1R3C) were significantly decreased in the AnCg of MDD group. While somatostatin (SST) mRNA expression was increased in AnCg of BPD in both microarray experimental duplicates utilizing U95Av2 and U133A, the finding was not replicated by qRT-PCR (Table 17).
  • IN SITU HYBRIDIZATION
  • We performed in situ hybridization for GPR37 using. GPR37 mRNA is preferentially expressed in subcortical white matter. GPR37 expression in the deeper layers (V-VI) is relatively higher than the superficial layers (I-III). GPR37 mRNA expression in subcortical white matter was higher in AnCg of the BPD subjects compared to the control subjects. GPR37 mRNA expression was rarely detected in AnCg of the MDD subjects analyzed. Figure 27 shows the dysregulation of genes involved in cAMP- and phosphatidylinositol signaling pathways in brain tissue from patients with BPD and MDD.
  • GENE EXPRESSION CHANGES IN AMYGDALA, HIPPOCAMPUS, NUCLEUS ACCUMBENS OF BIPOLAR DISORDER AND MAJOR DEPRESSIVE DISORDER.
  • We applied the same experimental design on amygdala, hippocampus and nucleus accumbens of BPD and MDD. Table 18 summarizes genes which are differentially expressed in amygdala, hippocampus and nucleus accumbens of BPD patients. Table 19 summarizes genes differentially expressed in the three brain regions of MDD.
  • Example 6.
  • This Example shows gene dysregulation in pathways related to Mitochondria, Proteasome, Apoptosis, and Chaperone in Mood Disorder. Three brain regions were studied: AnCg, Cerebellum, and DLPFC. The results are compiled in Tables 20-22.
  • Example 7 NCAM1 ASSOCIATION WITH BIPOLAR DISORDER AND SCHIZOPHRENIA AND SPLICE VARIANTS OF NCAM1.
  • SNP and sample selection: Genomic DNA (gDNA) was extracted from human postmortem brain cerebellum tissue. Primers were designed for SNP 9 and then tested via PCR to determine correct band size. Using the SNP 9 primers, gDNA of 40 cases (20 controls, 9 BPDs and 11 MDDs) was sequenced with both the forward and reverse primers. The SNPs were located in exons a, b and c. SNPs b and c are intronic and found just before exon 'b' (7 bps upstream) and 'c' (12 bps upstream) respectively. Exon 'a' did not have a SNP in close proximity.
  • The genotypes were collected on an additional 26 bipolar genomic DNA samples extracted from lymphocytes from the National Institute of Mental Health (NIMH) for all 4 SNPs: SNP 6, SNP 9, SNP b and SNP c (see Figure 26). A third cohort was genotyped consisting of the Stanley Foundation 105 dorsalateral prefrontal cortex (DLPFC) microarray samples (n = 35 controls, n = 35 bipolar disorder, n = 35 schizophrenia) (Table 24). The Stanley samples were genotyped for SNP 9 and SNP b. For final analysis, the three groups of bipolar cases were combined and three control groups were merged and used for statistical comparisons of SNP 9 and SNP b. The results show an association of SNP 9 and SNP b haplotype with bipolar disorder and schizophrenia.
  • GENOTYPE X DIAGNOSIS INTERACTION FOR NCAM1 SPLICE VARIANTS.
  • The splice variants are alternative splicing combinations of 3 mini-exons (a,b,c) with the fourth SEC exon are shown in Figure 30. Two mood disorders were tested (Bipolar Disorder, Type I and Major Depressive Disorder, Recurrent) and both showed differences in NCAM1 splice variants in the DLPFC.
  • The present data relates NCAM1 polymorphic variation to bipolar disorder and splice variations in mRNA occurring near the polymorphisms. A genotypic association between SNP b in NCAM1 and bipolar disorder and a suggestive association of SNP 9 with schizophrenia were found. Three of the two marker haplotypes for SNP 9 and SNP b, CT, C(T/C), and (C/A)(T/C) display varying frequency distribution between bipolar and controls. Schizophrenia and controls show differences in frequency distribution in four of the two marker haplotypes of SNP 9 and SNP b, CT, C(T/C), (C/A)(T/C) and (C/A)C. Bipolar disorder differs from schizophrenia for SNP 9 and SNP b by haplotype frequency differences. SNP b and SNP 9 are not in LD and they are individually related to schizophrenia (SNP 9) and bipolar disorder (SNP b).
  • The splice variant evidence for SNP 9 and b confirm that each SNP can be associated with differences in SEC exon splicing, thus providing some differential mechanisms for release of NCAM1 in the brain. We observed notable differences between polymorphisms in NCAM1 and the relative isoform variants of the SEC exon which can lead to truncation and secretion of NCAM1 in the brain. This finding concerning the difference in splice variant relative amounts as a function of certain genotypes was shown in three of the four SNPs where at least one genotype showed a difference in the amount of SEC by splice variant. This evidence suggests that the amount of SEC in brain is not regulated by just one genotype. Since the haplotypes composed of SNP 9 and SNP b are significantly different between controls and bipolar and between controls and schizophrenia this may support the observation of differential splicing patterns of the SEC exon found across many samples. Additionally, SNP 9 and SNP b are not in LD and thus the individual associations in schizophrenia and bipolar with these SNPs also may be transmitted through differential splicing patterns. The SEC exon was clearly regulated by certain combinations of mini-exons. We have identified discrete splice variants that can be further studied and are perhaps associated with regulatory intronic SNPs.
  • Example 8
  • Lithium has long been the drug of choice for treating manic-depressive illness (manic-depression; bipolar affective disorder, BPD). This Example shows non-human primate genes which exhibit differential expression in response to treatment with lithium. The results have implications for understanding the mood stabilizing effects of lithium in patients with manic depression.
  • Gene expression profiling was carried out on the anterior cingulate cortex (AnCg) using high-density oligonucleotide microarrays (Affymetrix GeneChips). We determined differential gene expression profiles of postmortem brains from lithium-treated healthy monkeys over those of untreated controls, and validated candidate genes against those known to be lithium-responsive or disease-selective.
  • Some of the candidate genes that responded to chronic lithium treatment were the same as those found with changed expression levels in postmortem brains of subjects with mood disorders. Our results show that the GSK3B signaling system is altered in BPD and that it is a physiological target of lithium. The observed GSK3B signaling system change thus constitutes an endophenotype that is likely to be common to BPD and schizophrenia, notwithstanding their clinical and phenotypic disparity. The results, by facilitating reconstruction of the genetic networks underlying BPD pathophysiology will facilitate the rational mood stabilizers targeting the signal transduction network via GSK3.
  • Major depressive disorder (MDD) and bipolar affective disorder (BPD) are two most severe mood disorders. MDD is characterized by clinical depression, while BPD is marked by recurrent and dramatic swings of emotional highs (mania) and lows (depression). For decades, lithium carbonate (Li2CO3, commonly known as "lithium") has been the benchmark medication for mania (hyperactive, incoherent and delusional behavior). Lithium unlike other anti-manic treatment agents is unique in its ability to abort the manic condition and restore patient's balanced mental status. Although numerous hypotheses have been proposed accounting for the neuro-protective properties of lithium, the precise molecular mechanism(s) by which lithium elicits its "mood stabilizing" effects in manic-depressive patients remains obscure. We addressed the challenge using a microarray strategy because it can permit simultaneous detection of multiple lithium-responsive genes and pathways in drug-treated primates and direct comparison of the observed gene expression changes with those found in postmortem brains from subjects with BPD. Lithium carbonate suspension (Roxane Laboratories, Inc., Columbus, OH) diluted in fruit juice was administered orally (18mg/kg body weight) to a targeted plasma level of 0.6 to 1.2 mgEq/mL. The animals received the drug twice a day for varying periods ranging from 4 months to 1 year and 5 months to circumvent their tendency to spit the drug out.
  • AnCg showed a total of 220 candidate transcripts (65 upregulated and 155 down-regulated). The candidate genes from AnCg are listed in Table 28. Ontological annotations mapped candidate genes to several different biological processes and pathways, including GSK3B signaling system, as predicted, in the AnCg.
  • AnCg involvement demonstrated in this study together with published reports of hippocampus involvement in lithium response implicates likely involvement of the limbic system in mood disorders as well as in the differential gene expression elicited by chronic lithium treatment. The present study also illuminates multiple interrelated functional networks of neuronal signaling pathways acting in unity with GSK3B as a pivotal functional switch regulating gene expression in behavioral diseases of apparent disparate phenotypic diversity, BPD and SZ. The results show that manipulating GSK3B could affect one or more of the inositol triphosphate, NF-kB family, mitochondrial apoptosis, and ubiquitin-proteasome pathways.
  • Example 9
  • V-ATPASE SUBUNITS AS GENE CANDIDATES OF INTEREST FOR MAJOR DEPRESSIVE DISORDER (MDD).
  • Of the 14 V-ATPase subunits that we have interrogated with Affy microarrays and Illumina microarrays, 7 subunits (50%) are differentially expressed (P<0.05) in hippocampal MDD versus control on either the Affymetrix or Illumina arrays. Three of the 7 subunits are differentially expressed in MDD hippocampus on both the Affymetrix and Illumina arrays (see Table 29). Two of the V-ATPase subunits are also differentially expressed (P<0.05) in our Affy microarray study of monkey hippocampus (i.e., Table 30, showing chronic social stress versus no-stress comparison). These findings demonstrate that drugs now being developed to inhibit V-ATPase in patients with cancer and osteoporosis may also prove useful as novel antidepressants.
  • The above examples are provided to illustrate the invention but not to limit its scope. Other variants of the invention will be readily apparent to one of ordinary skill in the art and are encompassed by the appended claims. All publications, databases, Genbank sequences, patents, and patent applications cited herein are hereby incorporated by reference.
    Figure imgb0001
    Figure imgb0002
    Table 2. Microarray data for all FGF transcripts reliably detected in either DLPFC or AnCg and summary data for confirmation studies.
    UniGene ID Transcript DLPFC AnCg
    p-value direction p-value direction
    Hs.278954 FGF1 <0.01‡,† Decreased 0.01 Decreased
    Hs.284244 FGF2 NS <0.01*,‡ Decreased
    Hs.433252 FGF7 NS NS
    Hs.111 FGF9 <0.01 Increased <0.01 * Increased
    Hs.343809 FGF12 NS <0.01* Increased
    Hs.6540 FGF13 NS NS
    Hs.223851 FGF14 0.05 Increased NS
    Hs.748 FGFR1 NS Increased NS
    Hs.404081 FGFR2 <0.01*,‡,† Decreased <0.01*,‡ Decreased
    Hs.1420 FGFR3 <0.01*,‡,† Decreased <0.01 * Decreased
    *Met FDR multiple testing correction at the level of accepting 5% false positives.
    Observation was confirmed in an independent cohort of MDD and control subjects given in Table 1b, meeting p-values of <0.05 in all cases indicated.
    Observation was confirmed by real-time PCR analysis with p<0.05.
    NS = not significant.
    Table 3. BPD
    UniGene ID Gene Symbol Gene Name RefSeq ID Gene Bank Acc. No. LocusLink Chromosome AriCg-BP AnCg-MD Criteria DLPFC-BP DLPFC-MD Criteria
    Hs.407520 CHN2 chimerin (chima erin) 2 NM_004 067 U07223 1124 Chr:7p15.3 UP NC 2a, 3
    Hs.13351 LANCL1 LanC lantibiotic synthetase component C-ike 1 (bacterial) NM_006 055 Y11395 10314 Chr:2q33-q35 UP NC 1, 2a UP NC 2a
    Hs.309090 SFRS7 splicing factor, arginine/serine rich 7, 35kDa NM_006 276 L41887 6432 Chr:2p22.1 UP NC 1, 2a, 3
    Hs.7910 RYBP RING1 and YY1 binding protein NM_012 234 AL049940 23429 Chr:3p14.2 UP NC 1, 2a, 3
    Hs.150101 LAMP1 lysosomal-associated membrane protein 1 NM_005 561 J04182 3916 Chr:13q34 UP NC 2a, 3
    Hs.90458 SPTLC1 serine palmitoyl transferase, Ion g chain base subunit 1 NM_006 415 Y08685 10558 Chr:9q22.2 UP NC 1, 2a, 3
    Hs.406532 RPN2 ribophorin II NM_002 951 AL031659 6185 Chr:20q12-q1 3.1 UP NC 2a UP NC 1, 2a
    Hs.408883 SCN1 B sodium channel , voltage-gated, type I, beta NM_001 037 L10338 6324 Chr:19q13.1 DOWN NC 1,3
    Hs.91971 CGEF2 cAMP-regulated guanine nucleotide exchange factor II NM_007 023 U78516 11069 Chr:2q31-q32 DOWN NC 1,3
    Hs.49117 --- hypothetical pro tein DKFZp564 N1662 --- AL080093 --- --- UP NC 1,3
    Hs.84244 KCNB1 potassium volta NM_004 L02840 3745 Chr:20q13.2 DOWN NC 1,3
    Table 4. MDD
    UniGene ID Gene Symbol Gene Name RefSeq ID Gene Bank Ace. No. Locus Link Chromosome AnCg-BP AnCg-MD Criteria DLPFC-BP DLPFC-MD Criteria
    Hs.5462 SLC4A 4 solute carrier family 4, sodi um bicarbonate cotransporter, member 4 NM_003759 AF007216 8671 Chr:4q21 NC DOWN 2b NC DOWN 3
    Hs.44 PTN pleiotrophin (heparin bindin g growth factor 8, neurite growth-promoting factor 1) NM_002825 M57399 5764 Chr:7q33 -q34 NC DOWN 1, 2b
    Hs.144845 BBOX1 butyrobetaine (gamma), 2-oxoglutarate dioxygenase ( gamma-butyrobetaine hydroxylase) 1 NM_003986 AF082868 8424 Chr:11p1 4.2 NC DOWN 1, 2b, 3
    Hs.170133 FOXO1 A forkhead box O1A (rhabdo myosarcoma) NM_002015 AF032885 2308 Chr:13q1 4.1 NC DOWN 1, 2b NC DOWN 1,3
    Hs.62192 F3 coagulation factor III (thro mboplastin, tissue factor) NM_001993 J02931 2152 Chr:1p22 -p21 NC DOWN 1,2b,3
    Hs.166994 FAT FAT tumor suppressor homolog 1 (Drosophila) NM_005245 X87241 2195 Chr:4q34 -q35 NC DOWN 1,2b,3
    Hs.82002 EDNRB endothelin receptor type B NM_000115 S57283 1910 13q22 NC DOWN 1,2b NC DOWN 1,2b, 3
    Hs.403997 VIL2 villin 2 (ezrin) NM_003379 X51521 7430 Chr:6q25. 2-q26 NC DOWN 1, 2b
    Hs.8022 TU3A TU3A protein NM_007177 AF035283 11170 Chr:3p21. 1 NC DOWN 1, 2b
    Hs.356876 GPR12 G protein-coupled receptor XM_291111 AK027494 16664 4p15.32- NC DOWN 1, 2b, 3
    5 125 7 p15.31
    Hs.450919 GPC5 glypican 5 NM_004466 U66033 2262 Chr:13q3 2 NC DOWN 1, 2b
    Hs.414151 DAAM2 dishevelled associated acti vator of morphogenesis 2 NM_015345 AB002379 23500 Chr:6p21. 1 NC DOWN 2b NC DOWN 3
    Hs.172089 PORIM IN pro-oncosis receptor inducing membrane injurygene NM_052932 AL050161 11490 8 Chr:11q2 2.1 NC DOWNY 1,3
    Hs.77546 ANKRD 15 ankyrin repeat domain 15 NM_015158 D79994 23189 Chr:9p24. 3 NC DOWN 1,3
    Hs.26208 COL16 A1 collagen, type XVI, alpha 1 NM_001856 M92642 1307 Chr:1p35 -p34 NC DOWN 1,3
    Hs.434494 SYNJ2 synaptojanin 2 MM_003898 AF039945 8871 Chr:6q25. 3 NC DOWN 1,3
    Hs.434418 MYT1 L myelin transcription factor 1-like --- AB029029 23040 Chr:2p25. 3 NC UP 1,3
    Hs.78748 RIMS3 regulating synaptic membr ane exocytosis 3 --- D87074 9783 Chr:1 pter -p22.2 NC UP 1,3
    Hs.436987 ZNF28 8 zinc finger protein 288 NM_015642 AL050276 26137 Chr:3q 13. 2 NC DOWN 1 NC DOWN 1
    Hs.391392 ID4 inhibitor of DNA binding 4, dominant negative helix-lo op-helix protein NM_001546 AL022726 3400 Chr:6p22 -p21 NC DOWN 1
    Hs.109052 C14orf 2 chromosome 14 open read ing frame 2 NM_004894 AF054175 9556 Chr:14q3 2.33 NC UP 1
    Hs.33455 PAD12 peptidyl arginine deiminase , type II NM_007365 AB023211 11240 Chr:1p35. 2-p35.1 NC DOWN 1
    Hs.438240 ZFYVE 16 zinc finger, FYVE domain containing 16 NM_014733 AB002303 9765 Chr:5p15. 2-q14.3 NC DOWN 1
    Hs.75462 BTG2 BTG family, member 2 NM_006763 U72649 7832 Chr:1 q32 NC DOWN 1
    Table 5: Growth Factor Pathway Genes
    UniGene ID Gene Symbol Gene Name RefSeq Transcript D I LocusLink Chromosomal Location %P DLPFCMD direction DLPFCBP direction AnCgMD direction AnCgBP direction
    Hs.433326 IGFBP2 insulin-like growth factor binding protein 2 36kDa NM_000597 3485 Chr:2q33-q34 25 up none up down
    Hs.16512 OGFRL1 opioid growth factor receptor-like 1 NM_024576 79627 Chr:6q13 28 down none none down
    Hs.799 DTR diphtheria toxin receptor (heparin-binding epidermal growth factor-like growth facto NM_001945 1839 Chr:5q23 38 none up none none
    Hs.105689 LTBP2 latent transforming rowth factor beta bin ding protein 2 NM_000428 4053 Chr:14q24 42 none none none down
    Hs.289019 LTBP3 latent transforming growth factor beta bin ding protein 3 NM_021070 4054 Chr:11q12 43 none none none up
    Hs.376032 PDGFA platelet-derived growth factor alpha polyp eptide NM_002607/// NM _033023 5154 Chr:7p22 43 none none none down
    Hs.839 IGFALS insulin-like growth fa ctor binding protein, acid labile subunit NM_004970 3483 Chr:16p13.3 48 none none none down
    Hs.342874 TGF8R3 transforming growth factor, beta receptor III (betaglycan, 300k NM_003243 7049 Chr:1p33-p32 54 Da) none up none none
    Hs.404081 FGFR2 Fibroblast growth fact or receptor 2 (bacteri a-expressed kinase, keratinocyte growth f factor receptor, crani ofacial dysostosis 1, Crouzon syndrome, Pfeiffer syndrome, J ackson-Weiss syndr ome) NM_000141 /// NM 022969 /// NM_02 2970 /// NM_02297 1 /// NM_022972 /// NM_022973 /// NM 022974 /// NM_02 2975 /// NM 02297 6///NM_023028/// NM_023029 /// NM _023030 /// NM_02 3031 2263 Chr:10q26 55 down none down up
    Hs.433252 FGF7 fibroblast growth factor 7 (keratinocyte growth factor) NM_002009 2252 Chr:15q15-q2 1.1 59 none down none none
    Hs.67896 OGFR opioid growth factor receptor NM_007346 11054 Chr:20q13.3 59 none down none down
    Hs.446350 TGFBRA P1 transforming growth factor, beta receptor associated protein 1 NM_004257 9392 Chr:2q12.2 61 up up none none
    Hs.194208 FRS3 TGFB2 fibroblast growth factor receptor substrate 3 transforming NM_006653 10817 Chr:6p21.1 64 none down up down
    Hs.169300 growth factor, beta 2 NM_003238 7042 Chr:1q41 68 none none down none
    Hs.411881 GRB14 growth factor recept or-bound protein 14 NM_004490 2888 Chr:2q22-q24 69 none up up up
    Hs.1420 FGFR3 fibroblast growth factor receptor 3 (achondroplasia, thanatophoric dwarfism) NM_000142 /// NM _022965 2261 Chr:4p16.3 76 down none down none
    Hs.450230 IGFBP3 insulin-like growth factor binding protein NM_000598 3 3486 Chr:7p13-p12 76 none down none down
    Hs.308053 IGF1 insulin-like growth factor 1 (somatomedin C) NM_000618 3479 Chr:12q22-q2 3 81 down none down down
    Hs.419124 MET met proto-oncogene (hepatocyte growth actor receptor) NM_000245 f 4233 Chr:7q31 81 none up up none
    Hs.284244 FGF2 fibroblast growth factor 2 (basic) NM_002006 2247 Chr:4q26-q27 84 none up down none
    Hs.76473 IGF2R insulin-like growth factor 2 receptor NM_000876 3482 Chr:6q26 92 none none down down
    Hs.410037 CTGF connective tissue growth factor NM_001901 1490 Chr:6q23.1 93 down none none up
    Hs.274313 IGFBP6 insulin-like growth factor binding protein 6 NM_002178 3489 Chr:12q13 94 none none up down
    Hs.111 FGF9 fibroblast growth factor 9 (glia-activating factor) NM_002010 2254 2 Chr:13q11-q1 99 up up up none
    Hs.380833 IGFBP5 insulin-like growth factor binding protein 5 NM_000599 3488 Chr:2q33-q36 99 down none none down
    Hs.79095 EPS15 substrate epidermal growth factor receptor pathway 15 NM_001981 2060 Chr:1p32 100 none none none up
    Hs.2132 EPS8 epidermal growth factor receptor pathway NM_004447 substrate 8 2059 Chr:12q23-q2 4 100 down up down none
    Hs.278954 ; FGF1 fibroblast growth factor 1 (acidic) NM_000800 /// NM _013394 /// NM_03 3136 /// NM_03313 7 2246 Chr:5q31 100 down none down none
    Hs.343809 FGF12 fibroblast growth factor 12 NM_004113 /// NM 021032 2257 Chr:3q28 100 none none none none
    Hs.7768 FIBP fibroblast growth factor (acidic) intracellular binding protein NM_004214 /// NM _198897 9158 Chr:11q13.1 100 none none none up
    Hs.127842 3 HDGFRP hepatoma-derived grNM_016073 owth factor, related protein 3 50810 Chr:15q11.2 100 none none up none
    Hs.416959 HGS hepatocyte growth fa ctor-regulated tyrosine kinase substrate NM_0.04712 9146 Chr:17q25 100 none none none up
    Hs.239176 IGF1R insulin-like growth factor 1 receptor NM_000875 3480 Chr:15q25-q2 6 100 none none none down
    Hs.435795 IGFBP7 insulin-like growth fa ctor binding protein 7 NM_001553 3490 Chr:4q12 100 down down down none
    Hs.439109 NTRK2 neurotrophic tyrosine kinase, receptor, type2 NM_006180 4915 Chr:9q22.1 100 down none down none
    ., Hs.26776 NTRK3 neurotrophic tyrosine kinase, receptor, type3 NM_002530 4916 Chr:15q25 100 none none none down
    Hs.43080 PDGFC platelet 3 derived grow platelet derived growth factor C NM_016205 56034 Chr:4q32 100 none none none up
    Hs.44 PTN pleiotrophin (heparin binding growth factor 8, neurite growth-pr omoting factor 1) /// pleiotrophin (heparin binding growth factor r 8, neurite growth-pr omoting factor 1) NM_002825 5764 Chr:7q33-q34 /// 100 down none down none
    Hs.114360 TSC22 transforming growth factor beta-stimutated protein TSC-22 NM_006022 /// NM _183422 8848 Chr:13q14- 100 none none up none
    Hs.73793 VEGF vascular endothelial growth factor NM_003376 7422 Chr:6p12 100 down none down none
    Figure imgb0003
    Figure imgb0004
    Figure imgb0005
    Figure imgb0006
    GROWTH FACTOR TABLE 7
    MDD I I BPD
    UniGene ID Gene Symbol Amy AnCg DLPFC HC nAcc Amy AnCg DLPFC HC nAcc
    Epidermal Growth Factor System
    Hs.419815 EGF -1.1
    Hs.79095 EPS 15 1.3
    Hs.147176 EPS15R
    Hs.2132 EPS8 -1.2
    Hs.799 DTR -1.1
    Fibroblast Growth Factor System
    Hs.278954 FGF1 -1.2 -1.4
    Hs.343809 FGF12 1.5 1.5
    Hs.6540 FGF13 1.5 1.5
    Hs.223851 FGF14 1.5 1.3 1.4
    Hs.284244 FGF2 -1.4 -1.3 -1.4 -1.8
    Hs.433252 FGF7 -1.1
    Hs.111 FGF9 1.4 1.2 1.3 1.6
    Hs.748 FGFR1
    Hs.404081 FGFR2 -1.1 -1.3 -1.2 -1.3
    Hs.1420 FGFR3 -1.2 -1.4 -1.5
    Hs.7768 FIBP 1.3
    Hs.194208 FRS3
    Insulin-Like Growth Factor System
    Hs.308053 IGF1
    Hs.239176 IGF1R -1.2 -1.3
    Hs.76473 IGF2R
    Hs.839 IGFALS
    Hs.433326 IGFBP2 1.4
    Hs.450230 IGFBP3 -1.3 -1.4
    Hs.1516 IGFBP4 -1.6
    Hs.380833 IGFBP5 -1.2 -1.2 -1.2
    Hs.274313 IGFBP6
    Hs.435795 IGFBP7
    Neurotrophins
    Hs.439027 BDNF
    Hs.439109 NTRK2 -1.2 -1.4 -1.3 -1.3
    Hs.26776 NTRK3
    Hs.194774 CNTFR
    Opioid Growth Factor System
    Hs.67896 OGFR
    Hs.16512 OGFRL 1 -1.2 -1.2
    Platelet-Derived Growth Factor System
    Hs.1976 PDGFB
    Hs.43080 PDGFC
    Hs.74615 PDGFR A -1.3 -1.3 -1.2 -1.3
    Hs.307783 PDGFR B
    Transforming Growth Factor System
    Hs.25195 EBAF
    Hs.435067 ECGF1
    Hs.170009 TGFA -1.1
    Hs.169300 TGFB2
    Hs.421496 TGFBI -1.9
    Hs.82028 TGFBR 2
    Hs.342874 TGFBR 3 -1.3
    Hs.446350 TGFBR AP1
    Hs.114360 TSC22
    Hs.241257 LTBP1 -1.2 -1.1
    Hs.105689 LTBP2 -1.2
    Hs.289019 LTBP3
    Vascular Endothelial Growth Factor System
    Hs.73793 VEGF -1.4
    Hs.78781 VEGFB
    Hs.79141 VEGFC
    Other Growth Factors/Receptors
    Hs.89525 HDGF
    Hs.127842 HDGFR P3 1.3 1.3 1.3 1.2
    Hs.44 PTN -1.2
    Hs.270833 AREG
    Hs.410037 CTGF
    Other Growth Factor Receptor Signaling Proteins
    Hs.512118 GRB 10 i
    Hs.411881 GRB14
    Hs.411366 GRB2 1.1
    Hs.416959 HGS 1.1
    Hs.419124 MET
    TABLE 8: GLU/GABA
    GABA/glutamate signaling genes in Mood Disorders -SFN genes For IDF inclusion
    BPD MDD
    UniGene ID Gene Common name Locus AnCg DLPFC HC Amy AnCg DLPFC HC Amy
    GABAergic:
    GABA A receptor
    Hs.24969 alpha 5 GABRA5 GABA (A) receptor, alpha 5 GABA-A-Ra5 15q11.2 1.58 1.64 1.58
    Hs.302352 beta 3 GABARB3 GABA (A) receptor, beta 3 GABA-A-Rb3 15q11.2 1.54
    Hs.7195 gamma 2 Glutamate receptor GABARG2 GABA (A) receptor, gamma 2 GABA-A-Rg2 5q3.1 1.58 1.64 1.59
    Hs.7117 ionotropic, AMPA1 GRIA1 IGluR1 5q31.1 1.18
    Hs.512145 Glutamate receptor, metabotropic 3 GRM3 Glutamate receptor, metabotropic 3 mGluR3 7q21.1 1.22 1.32
    Glutamate transporters
    Hs.349088 Glutamate transporter, Na+-dependent, EAAT2 SLC1A2 Solute carrier family 1 (glial high affinity GLT-1; EAAT2 11p13 0.84 0.71 0.87 0.81
    Hs.371369 Glutamate Transporter, Na+-dependent, EAAT1 SLC1A3 Solute carrier family 1 (glial high affinity - GLAST; EAAT1 5p13 0.65
    Glutamine synthetase
    Hs.442669 Glutamate-ammonia ligase GLUL Glutamate-ammonia ligase (glut synthetase) Glutamine synthetase 1q31 0.72 0.86
    GABA, gamma amino butyric acid; glut/ glutamate, glutamic acid; MDD, major depressive disorder; BPD, bipolar disorder
    AnCg, anterior cingulate cortex; DLPFC, dorso-lateral prefrontal cortex; HC, hippocampus; Amy, amygdala.
    Numbers (Fold Change) in RED denote INCREASES, and BLUE the DECREASES
    Figure imgb0007
    Figure imgb0008
    Figure imgb0009
    Figure imgb0010
    Figure imgb0011
    Figure imgb0012
    Figure imgb0013
    Figure imgb0014
    Figure imgb0015
    Figure imgb0016
    Figure imgb0017
    Figure imgb0018
    Figure imgb0019
    Figure imgb0020
    Figure imgb0021
    Figure imgb0022
    Figure imgb0023
    Figure imgb0024
    Figure imgb0025
    Figure imgb0026
    Figure imgb0027
    Figure imgb0028
    Figure imgb0029
    Figure imgb0030
    Figure imgb0031
    Figure imgb0032
    Figure imgb0033
    Figure imgb0034
    Figure imgb0035
    Figure imgb0036
    Figure imgb0037
    Figure imgb0038
    Figure imgb0039
    Figure imgb0040
    Figure imgb0041
    Figure imgb0042
    Table 12 Genes dysregulated in MD, BP, and schizophrenia
    Symbol Name UniGene ID AnCg DLPFC CB nAcc
    PTGDS Prostaglandin Hs.446429 CB-D nAcc-D
    PLAT Plasminogen activator, tissue Hs. 491582 AnCg-D DLPFC-D
    ADAMTS1 Disintegrin-like and metalloprotease Hs.534115 DLPFC-D nAcc-D
    Table 13
    Bipolar Disorder Major Depressive Disorder
    U95Av2 Cohort A U133A Cohort A U95Av2 Cohort A U133A Cohort A U133A Cohort B
    UniGene ID GDB Accession # Name Symbol Cytoband P Value %Fold Change P Value %Fold Change P Value %Fold Change P Value %Fold Change P Value %Fold Change
    Monoamine Metabolism
    Hs.370408 AL390148 Catechol-O-methyltransfera COMT 22q11.21-q11.23 < 0.01 15.8 < 0.01 11.5
    Hs.46732 NM_000898 Monoamine oxidase B MAOB Xp11.23 < 0.01 15.9 < 0.01 18.9
    Neuropeptide Ligand
    Hs.1832 BF680552 Neuropeptide Y NPY 7p15.1 < 0.01* 22.1 < 0.01* 33.0
    Hs.12409 BI918626 Somatostatin SST 3q28 < 0.01* 29.0 < 0.01* 22.1
    Hs.1408 BC053866 Endothelin 3 EDN3 20q13.2-q13.3 < 0.01 -12.3 < 0.01 -10.0
    GPCRs
    Hs.519057 L07615 Neuropeptide Y receptor Y1 NPY1R 4q31.3-q32 < 0.01* 24.4 < 0.01* 21.9 < 0.05 -17.7
    Hs.131138 NM_012344 Neurotensin receptor 2 NTSR2 2p25.1 < 0.01 -13.9 < 0.01 -18.6 < 0.01* -24.7
    Hs.112621 BC041407 Metabotropic Glutamate receptor 3 GRM3 7q21.1-q21.2 < 0.01* 33.4 < 0.01* 34.4
    Hs.88372 BC033742 Tachykinin receptor 2 TACR2 10q11-q21 < 0.01* -22.9
    Hs.154210 BC018650 Endothelial differentiation G-protein-coupled receptor 1 EDG1 1p21 < 0.01 -19.7 < 0.01* -21.2 < 0.01 -10.4
    Hs.126667 BC036034 Endothelial differentiation G-protein-coupled receptor 2 EDG2 9q31.3 < 0.05 24.1 < 0.01 18.2
    Hs.82002 NM_000115 Endothelin receptor type B EDNRB 13q22 < 0.01* -31.2 < 0.01 -15.9
    Hs.406094 BX649006 G protein-coupled receptor 37 GPR37 7q31 < 0.01* 53.1 < 0.01* 38.3 < 0.01* -53.5 < 0.01* -40.5 < 0.01* -27.0
    Hs.513633 NM_201524 receptor G protein-coupled 56 GPR56 16q13 < 0.01 -12.0 < 0.01 -15.9 < 0.01 -14.9
    Hs.148685 NM_016235 G protein-coupled receptor C-5-B GPRC5B 16p12 < 0.01* 38.4 < 0.01* 24.6 < 0.01* -36.5 < 0.01* -38.3 < 0.01* -30.3
    Hs.99195 XM_291111 G protein-coupled receptor 125 GPR125 4p15.31 < 0.05 -14.1 < 0.05 -11.5
    G protein and Regulators
    Hs.134587 BC026326 G protein alpha inhibiting activity polypeptide 1 GNAI1 7q21 < 0.01* 40.4
    Hs.24950 NM_003617 Regulator of G- protein signalling 5 RGS5 1q23.1 < 0.01 -19.0 < 0.01* -27.2
    Hs.368733 AK094559 Regulator of G- protein signalling 20 RGS20 8q12.1 < 0.01 15.6 < 0.01* -28.2 < 0.01* -29.2 < 0.01 -13,9
    Cyclic AMP Signaling Pathway
    Hs.416061 NM_005019 Phosphodiester ase 1A, calmodulin-dependent PDE1A 2q32.1 < 0.01* 20.1 < 0.01* 24.1
    Hs.9333 NM_173457 ase 8A Phosphodiester PDE8A 15q25.3 < 0.01 16.3 < 0.01 12.5 < 0.01* -24.3 < 0.01* -23.1 < 0.01 -17.8
    Hs.433700 NM_006823 cAMP-dependent Protein kinase inhibitor alpha PKIA 8q21.11 < 0.05 14.2 < 0.01 16.6
    Hs.183994 AK098311 Protein phosphatase 1, catalytic subunit, alpha PPP1CA 11q13 < 0.01 13.1 < 0.01 16.8
    Hs.303090 BX537399 Protein phosphatase 1, regulatory subunit 3C PPP1R3C 10q23-q24 < 0.01 -15.4 < 0.01* -35.4 < 0.01* -63.2 < 0.01* -21.4
    Hs.166071 AK026533 Cyclin-dependent CDK5 7q36 < 0.01 10.5 < 0.01 12.0
    Phosphatidylinositol Signaling Pathway
    Hs.374613 D26070 Inositol 1,4,5-triphosphate receptor, type 1 ITPR1 3p26-p25 < 0.05 13.0 < 0.01 17.4
    Hs.460355 AL833252 Protein kinase C, beta 1 PRKCB1 16p11.2 < 0.01 14.8 < 0.01 11.5
    Hs.478199 NM_002740 Protein kinase C, iota PRKCI 0 < 0.01* -29.5
    Hs.444924 NM_001263 CDP-diacylglycerol CDS1 4q21.23 < 0.01* -29.5
    Hs.32309 AK093560 kinase 1 polyphosphate-1-phosphatase INPP1 2q32 < 0.01* 21.5 < 0.01* 24.9
    Hs.369755 NM_014937 Inositol polyphosphate-5-phosphatase F INPP5F 10q26.11-q26.12 < 0.01* 21.8
    Hs.528087 NM_002221 Inositol 1,4,5-trisphosphate 3-kinase B ITPKB 0 < 0.01 14.8 < 0.05 11.5 < 0.01 -12.7 < 0.05 -12.3 < 0.01* -34.0
    Hs.175343 BX648778 Phosphoinositid e-3-kinase, class 2, alpha polypeptide PIK3C2A 11p15.5-p14 < 0.05 22.3 < 0.05 -14.1 < 0.01* -35.9
    Hs.497487 Y11312 Phosphoinositid e-3-kinase, class 2, beta polypeptide PIK3C2B 1q32 < 0.01 16.6 < 0.01 14.8
    Hs.132225 NM_181523 Phosphoinositide-3-kinase, regulatory subunit 1 PIK3RI 5q13.1 < 0.01* -32.0
    Hs.467192 AK090488 Protein phosphatase 2, regulatory subunit A, alpha isoform PPP2R1A 19q13.41 < 0.01 12.1 < 0.01 10.1
    Hs.146339 NM_002717 Protein phosphatase 2, regulatory subunit B, alpha isoform PPP2R2A 8p21.2 < 0.01* 32.3
    Table 14
    Bipolar Disorder Major Depressive Disorder
    U95Av2 Cohort A U133A Cohort A U95Av2 Cohort A U133A Cohort A U133A Cohort B
    Name Symbol UniGene ID GDB Acc # G protein Cytoband P Value %FC P Value %FC P Value %FC P Value %FC P Value %FC
    Monoamine Metabolism
    Catechol-O-methyltransferase COMT Hs.370408 AL390148 22q11.21- q11.23 < 0.01 15.8 < 0.01 11.5
    Monoamine oxidase MAOB B Hs.46732 NM_000898 Xp11.23 < 0.01 15.9 < 0.01 18.9
    Ligand peptide
    Neuropeptide Y NPY Hs.1832 BF680552 Gi, Gq 7p15 < 0.01* 22.1 < 0.01* 33.0
    Somatostatin SST Hs.12409 BI918626 Gi 3q28 < 0.01* 29.0 < 0.01* 22.1
    Endothelin 3 EDN3 Hs.1408 BC053866 Gq 20q13.2- q13.3 < 0.01 -12.3 < 0.01 -10.0
    GPCRs
    Neuropeptide Y receptor Y1 NPY1R Hs.519057 L07615 Gi 4q31-q32 < 0.01* 24.4
    Tachykinin receptor 2 TACR2 Hs.88372 BC033742 Gq 10q11-q21 < 0.01* -22.9
    Neurotensin receptor 2 NTSR2 Hs.131138 NM_012344 Gq 2p25.1 < 0.01 -13.9 < 0.01 -18.6 < 0.01* -24.7
    Endothelin receptor type B EDNRB Hs.82002 NM_000115 Gq 13q22 < 0.01* -31.2 < 0.01 -15.9
    Metabotropic Glutamate receptor 3 GRM3 Hs.112621 BC041407 Gi 7q21 < 0.01* 33.4 < 0.01* 34.4
    Endothelial differentiation GPCR 1 EDG1 Hs.154210 SC018650 Gi, G12 1p21 < 0.01 -19.7 < 0.01* -21.2 < 0.01 -10.4
    Endothelial differentiation GPCR 2 EDG2 Hs.126667 BC036034 Gi, Gq, G12 9q31.3 < 0.05 24.1 < 0.07 18.2
    G protein-coupled receptor 37 GPR37 Hs.406094 BX649006 Unknown 7q31 < 0.01* 53.1 < 0.01* 38.3 < 0.01* -53.5 < 0.01* -40.5 < 0.01* -27.0
    G protein-coupled receptor C-5-B GPRC5B Hs.148685 NM_016235 Unknown 16p12 < 0.01* 38.4 < 0.01* 24.6 < 0.01* -36.5 < 0.01* -38.3 < 0.01* -30.3
    G protein-coupled receptor 56 GPR56 Hs.513633 NM_201524 Unknown 16q13 < 0.01 -12.0 < 0.01 -15.9 < 0.01 -14.9
    G protein-coupfed receptor 125 GPR125 Hs.99195 XM_291111 Unknown 4p15 < 0.05 -14.1 < 0.05 -11.5
    Cyclic AMP Signaling Pathway
    G protein alpha inhibiting activity GNAI1 1 Hs.134587 BC026326 7q21 < 0.01* 40.4
    Regulator of G-protein signalling 20 RGS20 Hs.368733 AK094559 8q12 < 0.01* -28.2 < 0.01* -29.2 < 0.01 -13.9
    Phosphodiesterase 1A PDE1A Hs.416061 NM_005019 2q32 < 0.01* 20.1 < 0.01* 24.1
    Phosphodiesterase 8A PDE8A Hs.9333 NM_173457 15q25 < 0.01 16.3 < 0.01 12.5 < 0.01* -24.3 < 0.01* -23.1 < 0.01 -17.8
    Protein kinase A inhibitor alpha PKIA Hs.433700 NM_006823 8q21 < 0.05 14.2 < 0.01 16.6
    Cyclin-dependent kinase 5 CDK5 Hs.166071 AK026533 7q36 < 0.01 10.5 < 0.01 12.0
    Protein phosphatase 1, catalytic alpha PPP1CA Hs.183994 AK098311 11q13 < 0.01 13.1 < 0.01 16.8
    Protein phosphatase 1, regulatory 3C PPP1R3 C Hs.303090 BX537399 10q23-q24 < 0.01* -35.4 < 0.01* -63.2 < 0.01* -21.4
    Phosphatidylinositol Signaling Pathway
    Inositol polyphosphate-5-phosphatase A INPP5A Hs.523360 NM_005539 10q26 < 0.01 13.2 < 0.05 12.2
    Inositol polyphosphate-5-phosphatase F INPP5F Hs.369755 NM_014937 10q26 < 0.01* 21.8
    Inositol 1,4,5-trisphosphate 3-kinase B ITPKB Hs.528087 NM_002221 1q41-q43 < 0.01 14.8 < 0.05 11.5 < 0.01 -12.7 < 0.05 -12.3 < 0.01* -34.0
    Inositol poiyphosphafe-1-phosphatase INPP1 Hs.32309 AK093560 2q32 < 0.01* 21.5 < 0.01* 24.9
    CDP-diacylglycerol synthase 1 CDS1 Hs.444924 NM_001263 4q21.23 < 0.01* -29.5
    Phosphoinositide- 3-kinase catalytic 2A PIK3C2A Hs.175343 BX648778 11p15-p14 < 0.05 -14.1 < 0.01* -35.9
    Phosphoinositide- 3-kinase catalytic 2B PIK3C2B Hs.497487 Y11312 1q32 < 0.01 16.6 < 0.01 14.8
    Phosphoinositide- 3-kinase regulatory 1 PIK3R1 Hs.132225 NM_181523 5q13 < 0.01* -32.0
    Protein kinase C iota PRKCI Ns.478199 NM_002740 3p25-q27 < 0.01* -29.5
    Inositol 1,4,5-triphosphate receptor 1 ITPR1 Hs.374613 D26070 3p26-p25 < 0.05 13.0 < 0.01 17.4
    Protein kinase C beta 1 PRKCB1 Hs.460355 AL833252 16p11 < 0.01 14.8 < 0.01 11.5
    Table 15
    Bipolar Disorder Major Depresive Disorder
    U95Av2 Cohort A U133A Cohort A U95Av2 Cohort A U133A Cohort A U133A Cohort B
    Name Symbol UniGene ID GDB Accession # G protein Cytoband P Value %FC P Value %FC P Value %FC P Value %FC P Value %FC
    Protein phosphatase 1 regulatory 3C PPP1R3C Hs.303090 BX537399 10q23-q24 < 0.01* -26.4 < 0.01* -62.5 < 0.01* -29.6
    Phosphodiesterase 8A PDE8A Ns.9333 NM_173457 15q25 < 0.01* -32.0
    Inositol 1,4,5-trisphosphate 3-kinase B ITPKB Hs.528087 AJ242780 1q42 < 0.01 -12.5 < 0.01 * -46.1
    G protein beta 5 GNB5 Hs.155090 AK092059 15q21 <0.05 12.8 < 0.01 18.7
    Somatostatin SST Hs.12409 BI918626 Gi 3q28 < 0.01 -16.8 < 0.01* -25.7 < 0.01* -26.6 < 0.01* -34.2
    Adrenergic, beta-1-, receptor ADRB1 Hs.99913 BC045633 Gs 10q24-q26 < 0.01 -18.4 <0.05 -13.4
    Glutamate receptor, metabotropic 3 GRM3 Has. 112621 BC041407 Gi 7q21 < 0.01* 28.4
    G protein-coupled receptor C-5-B GPRC5B Hs.148685 NM_016235 Unknown 16p12 < 0.01* 31.8 < 0.01* -35.6 < 0.01* -34.3 < 0.01* -24.0
    G protein-coupled receptor 37 GPR37 Hs.406094 BX649006 Unknown 7q31 < 0.01* -62.5
    G protein-coupled receptor 56 GPR56 Hs.513633 NM_201524 Unknown 16q13 < 0.01 -14.2 < 0.01* -20.0 < 0.01* -23.2
    Table 16
    BPD MDD
    U95Av2 U95Av2
    Name Symbol UniGene ID GDB Accession # G protein Cytoband P Value %FC P Value %FC
    Phosphodiesterase 4B PDE4B Hs.198072 CR749667 1p31 < 0.01* -45.3
    Inositol polyphosphate-5-phosphatase A INPP5A Hs.523360 NM_005539 10q26 < 0.01* 26.1
    Regulator of G-protein signalling 20 RGS20 Hs.368733 AK094559 8q12 < 0.01* -23.8
    Proenkephalin PENK Hs.339831 AK091563 Gi 8q23-q24 < 0.01* 83.4 < 0.01* 34.5
    G protein-coupled receptor C-5-B GPRC5B Hs.148685 NM_016235 Unknown 16p12 <0.01* -31.7
    G protein-coupled receptor 37 GPR37 Hs.406094 BX649006 Unknown 7q31 < 0.01* -42.4
    Table 17
    BPD vs Control MDD vs Control
    UniGene Gene Name Symbol U95Av2 %FC U133A %FC qRT-PCR %FC U95Av2 %FC U133A %FC qRT-PCR %FC
    Hs.1832 Neuropeptide Y NPY 22.1 ** 33.0 ** 37.6 *
    Hs.12409 Somatostatin SST 29.0 ** 22.1 ** N.S.
    Hs.148685 G protein-coupled receptor C-5-B GPRC5B 38.4 ** 24.6 ** 46.8 * -36.5 ** -38.3 ** -54.0 **
    Hs.406094 G protein-coupled receptor 37 GPR37 53.1 ** 38.3 ** 54.1 * -53.5 ** -40.5 ** -62.7 *
    Hs.368733 Regulator of G-protein signalling 20 RGS20 -28.2 ** -29.2 ** -36.9 **
    Hs.303090 Protein phosphatase 1 regulatory subunit 3C PPPIR3C -35.4 ** -63.2 ** -46.1 **
    Hs.32309 Inositol polyphosphate-1-phosphatase INPP1 21.5 ** 24.9 ** 21.7 *
    Table 18
    BP - Control
    Amy 133A-22 HC 133A-22 nAcc 133A-22
    Name Symbol UniGene iD UGRepAcc Cytoband p-value %FC p-value %FC p-value %FC
    Ligands
    Adrenomedullin ADM Hs.441047 CR603703 11p15 <0.01* 31.53
    Brain-specific angiogenesis inhibitor 3 BAI3 Hs.13261 AB011122 6q12 <0.01* 22.18 <0.01* 36.44
    Cholecystokinin CCK Hs.458426 BC028133 3p22-p21 <0.01* 58.95 <0.01* 75.64
    Somatostatin SST Hs.12409 B1918626 3q28 <0.01* 43.64
    Chemokine (C-C motif) ligand 25 CCL25 Hs.310511 CR603063 19p13 <0.01* -23.50
    Chemokine (C-X-C motif) liqand 14 CXCL14 Hs.483444 NM_004887 5q31 <0.01* -21.63 <0.01* -37.40 <0.01* -26.52
    Frizzled homolog 7 (Drosophila) FZD7 Hs.173859 AB017365 2q33 <0.01* 26.17
    Monoamine oxidase B MAOB Hs.46732 NM_000898 Xp113 <0.01* 21.95
    Neuropeptide Y NPY Hs.1832 BF680552 7p15 <0.01* 27.35
    Neurotensin NTS Hs.80962 BF698911 12q21 <0.01* 31.10
    Prodynorphin PDYN Hs.22584 BC026334 20pter-p12 <0.01* 73.04 <0.01* 20.40
    Proenkephalin PENK Hs.339831 AK091563 8q23-q24 <0.01* 121.58 <0.01 19.08
    GPCR
    Neuropeptide Y receptor Y1 NPY1R Hs.519057 L07615 4q31-q32 <0.01* 42.45
    Gamma-aminobutyric acid (GABA) A receptor, delta GABRD Hs.113882 NM_000815 1p <0.01* 27.99
    Neurotensin receptor 2 NTSR2 Hs.131138 NM_012344 2p25 <0.01* -26.66
    Oxytocin receptor OXTR Hs.2820 NM_000916 3p25 <0.01* -27.98
    Adenosine A2a receptor ADORA2A Hs.197029 BC013780 22q11 <0.01* 42.45
    Cadherin, EGF LAG seven-pass G-type receptor 2 CELSR2 Hs.57652 AF234887 1p21 <0.01 * 20.09
    G protein-coupled receptor 116 GPR116 Hs.362806 BC066121 6p12 <0.01* -33.15
    G protein-coupled receptor 125 GPR125 Hs.99195 XM_291111 4p15 <0.05 -11.08
    G protein-coupled receptor 17 GPR17 Hs.46453 AK126849 2q21 <0.05 8.77
    G protein-coupled receptor 22 GPR22 Hs.432557 AK122621 7q22-q31 <0.05 12.47
    G protein-coupled receptor 37 GPR37 Hs.406094 BX649006 7q31 <0.01 -16.70
    G protein-coupled receptor 51 GPR51 Hs.198612 AF056085 9q22-q22 <0.01 -19.60 <0.01 15.51 <0.01* 23.38
    G protein-coupled receptor 6 GPR6 Hs.46332 NM_005284 6q21 <0.01* 78.20 <0.01 * 24.14
    G protein-coupled receptor, C- 5- B GPRC5B Hs.148685 NM_016235 16p12 <0.01 11.08
    Glutamate receptor, metabotropic 3 GRM3 Hs.112621 BC041407 7q21 <0.01* 28.68
    Histamine receptor H3 HRH3 Hs.251399 NM_007232 20q13 <0.01 18.90
    Dopamine receptor D1 DRD1 Hs.2624 NM_000794 5q35 <0.01* 53.37 <0.05 17.68
    Endothelin receptor type B EDNRB Hs.82002 NM_000115 13q22 <0.01* 24.03 <0.01* -37.40
    5-hydroxytryptamine (serotonin) receptor 2C HTR2C Hs.149037 NM_000868 Xq24 <0.01 * 97.85 <0.01 * 24.17 <0.01* 33.87
    G protein
    G protein, alpha inhibiting activity polypeptide 1 GNAI1 Hs.134587 BC026326 7q21 <0.01 * 22.98 <0.01 * 32.60
    Guanine nucleotide binding protein, beta polypeptide 1 GNB1 Hs.430425 AK123609 1p36 <0.01 19.19
    Guanine nucleotide binding protein (G protein), beta 5 GNB5 Hs.155090 AK092059 15q21 <0.01 19.68 <0.01* 21.92
    Guanine nucleotide binding protein (G protein), gamma 3 GNG3 Hs.179915 BM668891 11p11 <0.05 9.70 <0.01* 28.84 <0.01* 31.30
    Guanine nucleotide binding protein (G protein), gamma 7 GNG7 Hs.515544 AK024465 19p13 <0.01* 25.24
    Regulator of G protein signaling
    Regulator of G-protein signailing 1 RIGS1 Hs.75256 AK093544 1q31 <0.05 -9.16
    Regulator of G-protein signalling 2 RGS2 Hs.78944 BC042755 1q31 <0.01 * 23.56 <0.01* 20.60
    Regulator of G-protein signalling 20 of G-protein RGS20 Hs.368733 AK094559 8q12 <0.01 * 25.58
    Regulator of G-protein signalling 4 RGS4 Hs.386726 NM_005613 1q23 <0.05 -8.25 <0.01* 21.82
    Regulator of G-protein signalling 5 RGS5 Hs.24950 NM_003617 1q23 <0.01* -26.85 <0.01* -32.51 <0.01* -20.65
    Regulator of G-protein signalling 7 RGS7 Hs.130171 CR627366 1q43 <0.01 18.39
    Regulator of G-protein signalling 9 RGS9 Hs.132327 BC022504 17q23-q24 <0.01* 25.58
    Cyclic AMP signaling
    Protein kinase, cAMP-dependent, catalytic, beta PRKACB Hs.487325 BX537705 1p36 <0.01* 26.80 <0.01* 20.88 <0.01* 22.95
    Protein kinase, cAMP-dependent, regulatory, type I, alpha PRKAR1A Hs.280342 CR749311 17q23-q24 <0.01* 21.71 <0.01* 23.64 <0.01* 26.15
    Protein kinase, cAMP-dependent, regulatory, type II, beta PRKAR2B Hs.433068 BC075800 7q22 <0.01* 23.30 <0.01* 54.89
    Protein kinase (cAMP-dependent, catalytic) inhibitor alpha PKIA Hs.433700 NM_006823 8q21 <0.01* 30.00
    Phosphodiesterase 8A PDE8A Hs.9333 NM_173457 15q25 <0.01* -25.56
    Cyclic AMP phosphoprotein, 19 kD ARPP-19 Hs.512908 AL833077 15q21 <0.01 * 27.29 <0.01 19.35
    Adenylate cyclase-associated protein, 2 CAP2 Hs.132902 NM_006366 6p22 <0.05 10.52 <0.01* 35.43
    Cyclin-dependent kinase 5 CDK5 Hs.166071 AK026533 7q36 <0.01* 20.85
    Phosphatidylinositol signaling
    Diacylglycerol kinase, beta 90kDa DGKB Hs.487619 NM_004080 7p21 <0.01 18.61 <0.01* 26.46
    Inositol polyphosphate-5-phosphatase INPP5A Hs.523360 NM_005539 10q26 <0.01* 34.39 <0.01* 27.86
    Inositol polyphosphate-5-phosphatase F INPP5F Hs.369755 NM_D14937 10q26 <0.01* 31.06 <0.01* 29.81
    Inositol 1,4,5-triphosphate receptor, type 1 ITPR1 Hs.374613 D26070 3p26-p25 <0.01* 23.50 <0.01* 29.24
    Phosphatidylinositol-4-phosphate 5-kinase, tvne I, beta PIP5K1B Hs.534371 BC030587 9q13 <0.01* 20.75
    Phosphatidylinositol-4-phosphate 5-kinase, type II, gamma PIP5K2C Hs.144502 AK125526 12q13 <0.01* 23.03
    Phospholipase C, beta 1 (phosphoinositide-specific) PLCB1 Hs.310537 NM_82734 20p12 <0.01 19.28
    Protein kinase C, beta 1 PRKCB1 Hs.460355 AL833252 16p11 <0.01* 23.66 <0.01 18.25
    Myristoylated alanine-rich protein kinase C substrate MARCKS Ns.519909 NM_002356 6q22 <0.01* 23.12
    Growth associated protein 43 GAP43 Hs.134974 AK091466 3q13 <0.01 * 27.99 <0.01* 46.79
    YWHA
    Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, beta polypeptide YWHAB Hs.279920 NM_003404 20q13 <0.01* 37.20 <0.01* 22.45
    Chromosome 22 open reading frame 24 YWHAH Hs.226755 CR622695 22q12 <0.01* 73.39 <0.01* 66.65
    Tyrosine 3-monoo3cygenase/tryptophan 5-monooxygenase activation protein, theta polypeptide YW HAQ Hs.74405 NM_006826 2p25 <0.01* 32.96
    Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide YWHAZ Hs.492407 BC051814 8q23 <0.01 13.55 <0.01* 42.25 <0.01 * 31.83
    Calcium signaling
    Calmodulin 1 (phosphorylase kinase, delta) CALM1 Hs.282410 BC047523 14q24-q31 <0.01* 21.80
    Calmodulin 3 (phosphorylase kinase, delta) CALM3 Hs.515487 AK094964 19q13 <0.01* 30.65
    Calcium/calmodulin-dependent protein kinase I CAMK1 Hs.434875 AK094026 3p25 <0.05 18.27
    Calcium/calmodulin-dependent protein kinase 11 alpha CAMK2A Hs.143535 NM_015981 5q32 <0.01* 20.41
    Calcium/calmodulin-dependent protein kinase II beta CAMK2B Hs.351887 NM_001220 22q12 <0.01* 21.93
    Calmodulin binding transcription activator 1 CAMTA1 Hs.397705 NM_015215 1p36 <0.01 16.05 <0.05 14.94
    Doublecortin and CaM kinase-like 1 DCAMKL1 Hs.507755 NM_004734 13q13 <0.01* 29.98
    MAPK signaling
    P21/Cdc42/Rac1-activated kinase 1 (STE20 homolog, yeast) PAK1 Hs.435714 NM_002576 11q13-q14 <0.05 13.71 <0.01 16.23
    Mitogen-activated protein kinase kinase 1 MAP2K1 Hs.145442 NM_002755 15q22 <0.01* 33.40 <0.01* 57.55 <0.01* 46.39
    Mitogen-activated protein kinase kinase 1 interacting protein 1 MAP2K1IP 1 Hs.433332 AK022313 4q23 <0.01* 20.94
    Mitogen-activated protein kinase kinase 4 MAP2K4 Hs.514681 AK131544 17p11 <0.01 17.47 <0.01* 29.05 <0.01* 22.97
    Mitogen-activated protein kinase kinase kinase kinase4 4 MAP4K4 Hs.431550 NM_145686 2q11-q12 <0.01* -23.32
    Mitogen-activated protein kinase 1 MAPK1 Hs.431850 NM_002745 22q11 <0.01* 31.80 <0.01* 52.27 <0.01* 41.35
    Mitogen-activated protein kinase 10 MAPK10 Hs.25209 AK124791 4q22-q23 <0.01* 25.47
    Mitogen-activated protein kinase 9 MAPK9 Hs.484371 BC032539 5q35 <0.01 13.93 <0.01* 22.88
    Protein Phosphatase
    Protein phosphatase 1, catalytic subunit, gamma isoform Protein phosphatase 1, regulatory (inhibitor) subunit 2 PPP1 CC Hs.79081 NM_002710 12q24 <0.01* 25.65
    PPP1R2 Hs.184840 NM_006241 3q29 <0.01* 21.53
    Protein phosphatase 1, regulatory subunit 3C PPP1R3C Hs.303090 BX537399 10q23-q24 <0.01 -16.80 <0.01* -47.55
    Protein phosphatase 2C, magnesium- PPM2C Hs.22265 NM_018444 8q22 <0.05 23.33
    dependent, catalytic subunit
    Protein phosphatase 2 (formerly 2A), catalytic subunit, alpha isoform PPP2CA Hs.483408 BX640662 5q23-q31 <0.01* 24.59
    Protein phosphatase 2 (formerly 2A), regulatory subunit A (PR 65), alpha isoform PPP2R1A Hs.467192 AK090488 19q13 <0.01* 27.34 <0.01* 24.11
    Protein phosphatase 2 (formerly 2A), regulatory subunit B (PR 52), beta isoform PPP2R2B Hs.193825 M64930 5q31-5q32 <0.01 * 38.94
    Protein phosphatase 2, regulatory subunit B (B56), gamma isoform PPP2R5C Hs.368264 NM_002719 14q32 <0.01* 23.54
    Protein phosphatase 3 (formerly 2B), catalytic subunit, alpha isoform (calcineurin A alpha) PPP3CA Hs.435512 NM_000944 4q21-q24 <0.01* 29.94 <0.01* 23.23
    Protein phosphatase 3 (formerly 2B), catalytic subunit, beta isoform (calcineurin A beta) PPP3CB Hs.500067 BC028049 10q21-q22 <0.01* 20.95 <0.01 * 45.82 <0.01* 31.17
    Protein phosphatase 3 (formerly 2B), regulatory subunit B, 19kDa, alpha isoform (calcineurin B, type I) PPP3R1 Hs.280604 BC027913 2p15 <0.01* 25.69
    AP1
    V-fos FBJ murine osteosarcoma viral oncogene homolog FOS Hs.25647 BX647104 14q24 <0.01* -40.86 <0.01* -80.23
    V-jun sarcoma virus 17 oncogene homolog (avian) JUN Hs.525704 NM_002228 1p32-p31 <0.01* -25.24
    Small G protein
    Rho guanine nucleotide exchange factor (GEF) 3 ARHGEF3 Hs.476402 AL833224 3p21-p13 <0.01* 31.85
    Ras homolog gene family, member I ARHI Hs. 194695 AK096393 1p31 <0.01 * 132.94 <0.01* 33.15
    DIRAS family, GTP-binding RAS-like 2 DIRAS2 Hs.165636 NM_017594 9q22
    RAB31, member RAS oncogene family RAB31 Hs.99528 NM_006868 18p11 <0.05 -9.44
    RAB33A, member RAS oncogene family RAB33A Hs.56294 AK094927 Xq25 <0.01* 21.72
    Rab acceptor 1 (prenylated) RABAC1 Hs.11417 BE779053 19q13 <0.01* 21.97
    RAN binding protein 6 RANBP6 His. 167496 BX537405 9p24 <0.01* 25.82
    RAP1, GTPase activating protein 1 RAP1GA1 Hs.148178 BC035030 1p36-p35 <0.01* 52.74
    Hypothetical protein LOC145899 RASGRF1 Hs.459035 NM_002891 15q24 <0.01 * 28.36
    RAS guanyl releasing protein 1 (calcium and DAG-regulated) RASGRP1 Hs.511010 AF081195 15q15 <0.01* 24.37
    RAS guanyl releasing protein 3 (calcium and DAG-regulated) RASGRP3 Hs.143674 BC027849 2p25-p24 <0.01 -14.27
    Ras-like without CAAX 2 RIT2 Hs.464985 AL713637 18q12 <0.01 * 32.56
    RAS-related on chromosome 22 RRP22 Hs.73088 NM_001007279 22q12 <0.01* 20.87
    V-Ki-ras2 Kirsten rat sarcoma 2 viral oncogene homolog KRAS2 Hs.505033 NM_033360 12p12 <0.01* 22.38
    Ras-GTPase activating protein SH3 domain- G3BP2 Hs.303676 NM_203505 4q21 <0.01* 23.35 <0.01* 36.80
    binding protein 2
    Potassium channel
    Potassium channel modulatory factor 1 KCMF1 HS.345694 MM_020122 2p11 <0.01 * 22.07
    Potassium voltage-gated channel, shaker-related beta 2 KCNAB2 Hs.440497 AK124696 1p36 <0.01 * 24.09
    Potassium voltage-gated channel, Shal-related 2 KCND2 Hs.21703 AB028967 7q31 <0.01* 20.05
    Potassium inwardly-rectifying channel, subfamily J 2 KCNJ2 Hs.1547 NM_000891 17q23-q24 <0.01 * -28.13
    Potassium inwardly-rectifying channel, subfamily J 6 KCNJ6 Hs.50927 AK058042 21q22 <0.01 * 26.12
    Potassium inwardly-rectifying channel, KCNJ13 Hs.467338 NM_002242 2q37 <0.01* 31.87
    subfamily J 13 Potassium channel, subfamily K, member 1 KCNK1 Hs.208544 AL833343 1q42-q43 <0.01* 23.33
    Potassium intermediate/small conductance calcium-activated channel, subfamily N, member 2 KCNN2 Hs.98280 NM_021614 5q22 <0.01 * 28.96
    Potassium intermediate/small conductance calcium-activated channel, subfamily N, Hs.490765 BX649146 1q21 <0.01 -13.90
    member 3 Potassium channel, subfamily V, member 1 KCNN3 KCNV1 Hs.13285 NM_014379 8q22-q24 <0.01 11.16
    Sodium channel Sodium channel, voltage-gated, type III, beta SCN3B Hs.4865 AB032984 11q24 <0.01* 41.71 <0.01* 37.41
    Solute carrier family 12, (potassium-chloride transporter) member 5 SLC12A5 Hs.21413 NM_020708 20q132 <0.01 11.53 <0.01* 21.22
    Solute carrier family 24 (sodium/potassium/calcium exchanger), SLC24A3 Hs.211252 AL833544 20p13 <0.01* 60.32
    member 3 Voltage-dependent anion channel 1 VDAC1 Hs.519320 AK122953 5q31 <0.01 * 27.48
    Calcium Channel
    Calcium channel, voltage-dependent, alpha 2/delta 3 CACNA2D3 Hs.369421 NM_018398 3p21 <0.01 * 36.34
    Calcium channel, voltage-dependent, beta 2 subunit CACNB2 Hs.59093 NM_000724 10p12 <0.01* 21.04 <0.01* 23.85
    Calcium channel, voltage-dependent, beta 3 subunit CACNB3 Hs.250712 AK1 22911 12q13 <0.01 * 22.11
    Calcium channel, voltage-dependent, gamma subunit 3 CACNG3 Hs.7235 AK095553 16p12-p13 <0.01 * 32.54
    Chloride channel
    Chloride intracellular channel 4 CLIC4 Hs.440544 AL117424 1p36 <0.05 -17.97
    Table 19
    MD - Control
    Amy 133A-22 HC 133A-22 nAcc 133A-22
    Name Symbol UniGene ID UGRepAcc Cytoband p-value %FC p-value %FC p-value %FC
    Ligands
    Adrenomedullin ADM Hs.441047 CR603703 11p15 <0.05 -7.16 <0.01* -34.24
    Brain-specific angiogenesis inhibitor 3 BAI3 Hs.13261 AB011122 6q12 <0.01* 20.13 <0.01* 20.61
    Cholecystokinin CCK Hs.458426 BC028133 3p22-p21 <0.01* 29.74 <0.01* 81.09
    Somatostatin SST Hs.12409 BI918626 3q28 <0.01* 20.70
    Frizzled homolog 7 (Drosophila) FZD7 Hs.173859 AB017365 2q33 <0.05 8.11
    Latrophilin 2 LPHN2 Hs.24212 AF104266 1p31 <0.01* 29.33
    Prodynorphin PDYN Hs.22584 BC026334 20pter-p12 <0.01* 34.43 <0.01* 31.55
    Proenkephalin PENK Hs.339831 AK091563 8q23-q24 <0.01* 57.92 <0.01* 21.80 <0.01* 22.13
    Prostaglandin D2 synthase 21 kDa (brain) PTGDS Hs.446429 BM805807 9q34-q34 <0.01 * -23.24
    GPCR
    Neuropeptide Y receptor Y1 NPY1 R Hs.519057 L07615 4q31-q32 <0.01* 44.09
    Gamma-aminobutyric acid (GABA) A receptor, delta GABRD Hs.113882 NM_000815 1p <0.01* 22.04 <0.01* 22.85
    Neurotensin receptor 2 NTSR2 Hs.131138 NM_012344 2p25 <0.01* -37.48 <0.01 -17.42
    Oxytocin receptor OXTR Hs.2820 NM_000916 3p25 <0.01 -15.90
    Cholecystokinin B receptor CCKBR Hs.203 AF239668 11p15 <0.01* 27.01
    Adenosine A2a receptor ADORA2A Hs.197029 BC013780 22q11 <0.01 18.30 <0.01* 23.23
    Angiotensin II receptor-like 1 AGTRL1 Hs.438311 AK075252 11q12 <0.01* -22.99
    G protein-coupled receptor 125 GPR125 Hs.99195 XM 291111 4p15 <0.01* -22.88
    G protein-coupled receptor 17 GPR17 Hs.46453 AK126849 2q21 <0.01* -27.08
    G protein-coupled receptor 22 GPR22 Hs.432557 AK122621 7q22-q31 <0.01* 24.51
    G protein-coupled receptor 37 GPR37 Hs.406094 BX649006 7q31 <0.01 * -43.90
    G-protein coupled receptor 37 like 1 GPR37L1 Hs.132049 BC050334 1q32 <0.01* -27.67
    G protein-coupled receptor 51 GPR51 Hs.198612 AF056085 9q22-q22 <0.01* 31.80 <0.01* 29.07
    G protein-coupled receptor 56 GPR56 Hs.513633 NM_201524 16q13 <0.01* -24.98
    G protein-coupled receptor 6 GPR6 Hs.46332 NM_005284 6q21 <0.01* 27.88 <0.01* 41.21
    Chemokine (C-X-C motif) receptor 4 CXCR4 Hs.421986 CR614663 2q21 <0.01* -20.68
    G protein-coupled receptor, C- 5- B GPRC5B Hs.148685 NM_016235 16p12 <0.01* -27.94 <0.01* -36.01
    Histamine receptor H3 HRH3 Hs.251399 NM_007232 20q13 <0.01* 30.42
    Dopamine receptor D1 DRD1 Hs.2624 NM_000794 5q35 <0.01* 27.79 <0.01* 25.88
    Endothelial differentiation, sphingolipid GPCR 1 EDG1 Hs.154210 BC018650 1p21 <0.01* -20.04
    Endothelin receptor type B EDNRB Hs.82002 NM_000115 13q22 <0.01* -64.83 <0.01 -14.45 <0.01 * -37.17
    5-hydroxytryptamine (serotonin) receptor 2A HTR2A Hs.424980 NM_000621 13q14-q21 <0.01* 45.03 <0.01* 42.32 -
    5-hydroxytryptamine (serotonin) receptor 2C HTR2C Hs.149037 NM_000868 Xq24 <0.01 14.27 <0.01 -18.49 <0.01* 31.98
    G protein
    Guanine nucleotide binding protein (G protein), alpha 13 GNA13 Hs.515018 NM 006572 17q24 <0.01* -20.84
    G protein, alpha inhibiting activity polypeptide 1 GNAI1 Hs. 134587 BC026326 7q21 <0.05 17.13
    Guanine nucleotide binding protein, beta polypeptide 1 GNB1 Hs.430425 AK123609 1p36 <0.01* 22.06
    Guanine nucleotide binding protein (G protein), beta 5 GNB5 Hs.155090 AK092059 15q21 <0.01* 24.99 <0.01* 32.65
    Guanine nucleotide binding protein (G protein), gamma 12 GNG12 Hs.431101 NM_018841 1p31 <0.01* -21.46
    Guanine nucleotide binding protein (G protein), gamma 3 GNG3 Hs.179915 BM668891 11p11 <0.01* 26.57 <0.01* 41.14 <0.05 14.55
    Regulator of G protein signaling
    Regulator of G-protein signalling 1 RGS1 Hs.75256 AK093544 1q31 <0.01* -34.56
    Regulator of G-protein signalling 2 RGS2 Hs.78944 BC042755 1q31 <0.01* 36.07 <0.01 17.15
    Regulator of G-protein signalling 20 RGS20 Hs.368733 AK094559 8q12 <0.01* -32.34
    Regulator of G-protein signalling 4 RGS4 Hs.386726 NM_005613 1q23 <0.01 * 72.22 <0.01 * 40.31 <0.05 10.50
    Regulator of G-protein signalling 5 RGS5 Hs.24950 NM_003617 1q23 <0.01 8.03
    Regulator of G-protein signalling 7 RGS7 Hs.130171 CR627366 1q43 <0.01 * 36.06 <0.01 * 36.42
    Regulator of G-protein signalling 9 RGS9 Hs.132327 BC022504 17q23-q24 <0.01 11.68
    Cyclic AMP signaling
    Protein kinase, cAMP-dependent, catalytic, beta PRKACB Hs.487325 BX537705 1p36 <0.01 17.60 <0.01 16.51
    Protein kinase, cAMP-dependent, regulatory, type I, alpha (tissue specific extinguisher 1) PRKAR1A Hs.280342 CR749311 17q23-q24 <0.01 11.92 <0.05 10.27
    Protein kinase, cAMP-dependent, regulatory, type II, beta PRKAR2B Hs.433068 BC075800 7q22 <0.01 * 26.41 <0.01* 52.17
    Protein kinase (cAMP-dependent, catalytic) inhibitor alpha PKIA Hs.433700 NM_006823 8q21 <0.05 14.78
    Phosphodiesterase 4D interacting protein (myomegalin) PDE4DIP Hs.487925 NM_014644 1q12 <0.01* -21.98
    Phosphodiesterase 8A PDE8A Hs.9333 NM_173457 15q25 <0.01* -20.78 <0.01* -39.39
    Phosphodiesterase 8B PDE8B Hs.78106 AF079529 5q13 <0.01* 20.36
    Cyclic AMP phosphoprotein, 19 kD ARPP-19 Hs.512908 AL833077 15q21 <0.01* 24.18 <0.01* 25.33 <0.01* 27.45
    Adenylate cyclase-associated protein, 2 CAP2 Hs.132902 NM_006366 6p22 <0.01* 32.38 <0.01* 27.21
    Cyclin-dependent kinase 5 CDK5 His.166071 AK026533 7q36 <0.01 18.67
    Phosphatidylinositol signaling
    Diacylglycerol kinase, beta 90kDa DGKB Hs.487619 NM_004080 7p21 <0.01* 32.70 <0.01* 26.36
    Inositol polyphosphate-5-phosphatase, 40kDa INPP5A Hs.523360 NM_005539 10q26 <0.01* 22.52 <0.01* 40.16 <0.01* 22.98
    Inositol polyphosphate-5-phosphatase F INPP5F Hs.369755 NM_014937 10q26 <0.01* 40.58 <0.01* 45.18 <0.01* 32.42
    Inositol 1,4,5-trisphosphate 3-kinase A ITPKA Hs.2722 BC026331 15q14-q21 <0.01* 29.49 <0.01* 32.18
    Inositol 1,4,5-trisphosphate 3-kinase B ITPKB Hs.528087 AJ242780 1q423 <0.01* -34.51 <0.01* -34.14
    Inositol 1,4,5-triphosphate receptor, type 1 ITPR1 Hs.374613 D26070 3p26-p25 <0.01* 45.62 <0.01* 51.25
    Phosphoinositide-3-kinase, class 2, alpha polypeptide PIK3C2A Hs.175343 BX648778 11p15-p14 <0.01* -63.06
    Phosphoinositide-3-kinase, class 2, alpha polypeptide PIK3C2A Hs.175343 BX648778 11p15-p14 <0.01* -63.06
    Phosphatidylinositol-4-phosphate 5-kinase, type I, beta PIP5K1B Hs.534371 BC030587 9q13 <0.01* 24.44
    Phosphatidylinositol-4-phosphate 5-kinase, type II, gamma PiP5K2C Hs.144502 AK125526 12q13 <0.05 17.13
    Phospholipase C, beta 1 (phosphoinositide-specific) PLCB1 Hs.310537 NM_182734 20p12 <0.01* 26.19
    Protein kinase C, beta 1 PRKCB1 Hs.460355 AL833252 16p11 <0.01* 51.20 <0.01* 43.94
    Growth associated protein 43 GAP43 Hs.134974 AK091466 3q13 <0.01* 49.55 <0.01* 30.12
    YWHA
    Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, beta polypeptide YWHAB Hs.279920 NM_003404 20q13 <0.01 * 20.42 <0.01* 30.24 <0.05 13.58
    Chromosome 22 open reading frame 24 YWHAH Hs.226755 CR622695 22q12 <0.01* 31.18 <0.01* 56.74 <0.01 * 26.27
    Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide YWHAZ Hs.492407 BC051814 8q23 <0.01 * 20.06 <0.01* 30.03 <0.01* 20.91
    Calcium signaling
    Calmodulin 1 (phosphorylase kinase, delta) CALM1 Hs.282410 BC047523 14q24-q31 <0.01 16.48
    Calmodulin 3 (phosphorylase kinase, delta) CALM3 Hs.515487 AK094964 19q13 <0.01 14.08
    Calcium/calmodulin-dependent protein kinase I CAMK1 Hs.434875 AK094026 3p25 <0.01* 20.75
    Calcium/calmodulin-dependent protein kinase II alpha CAMK2A Hs.143535 NM_015981 5q32 <0.01* 30.59
    Calcium/calmodulin-dependent protein kinase II beta CAMK2B Hs.351887 NM_001220 22q12 <0.05 13.91
    Calcium/calmodulin-dependent protein kinase II CaMKIINal pha Hs.197922 CR604926 1p36 <0.01 * 21.16
    Calcium/calmodulin-dependent protein kinase kinase 2, beta CAMKK2 Hs.297343 NM_006549 12q24 <0.01 * 22.16
    Calmodulin binding transcription activator 1 CAMTA1 Hs.397705 NM_015215 1p36 <0.01* 31.29 <0.01* 41.27
    Doublecortin and CaM kinase-like 1 DCAMKL1 Hs.507755 NM_004734 13q13 <0.05 16.77
    MAPK signaling
    P21/Cdc42/Rac1-activated kinase 1 (STE20 homolog, yeast) PAK1 Hs.435714 NM_002576 11q13-q14 <0.01* 32.71 <0.01* 36.58
    Mitogen-activated protein kinase kinase 1 MAP2K1 Hs.145442 NM_002755 15q22 <0.01* 23.61 <0.01* 35.94 <0.01* 26.19
    Mitogen-activated protein kinase kinase 1 interacting protein 1 MAP2K1IP 1 Hs.433332 AK022313 4q23 <0.05 13.15
    Mitogen-activated protein kinase kinase 4 MAP2K4 Hs.514681 AK131544 17p11 <0.01* 23.15 <0.01 19.77 <0.05 8.77
    Mitogen-activated protein kinase kinase kinase kinase 4 MAP4K4 Hs.431550 NM_145686 2q11-q12 <0.01* -24.08
    Mitogen-activated protein kinase kinase kinase kinase 5 MAP4K5 Hs.130491 NM_198794 14q11-q21 <0.01* -24.23
    Mitogen-activated protein kinase 1 MAPK1 Hs.431850 NM_002745 22q11 <0.05 9.57 <0.01* 22.13
    Mitogen-activated protein kinase 10 MAPK10 Hs.25209 AK124791 4q22-q23 <0.01* 21.09
    Mitogen-activated protein kinase 6 MAPK6 Hs.411847 NM_002748 15q21 <0.01* 23.03
    Mitogen-activated protein kinase 9 MAPK9 Hs.484371 BC032539 5q35 <0.01* 21.30 <0.05 12.07
    Protein Phosphatase
    Protein phosphatase 1, regulatory (inhibitor) subunit 2 PPP1R2 Hs.184840 NM_006241 3q29 <0.01* 20.02
    Protein phosphatase 1, regulatory subunit 3C PPP1R3C Hs.303090 BX537399 10q23-q24 <0.01* -54.15 <0.01* -47.47 <0.01* -43.72
    Protein phosphatase 2C, magnesium-dependent, catalytic subunit PPM2C Hs.22265 NM_018444 8q22 <0.01* 35.09 <0.01* 47.45
    Protein phosphatase 2 (formerly 2A), catalytic subunit, alpha isoform PPP2CA Hs.483408 BX640662 5q23-q31 <0.01 17.92
    Protein phosphatase 2 (formerly 2A), regulatory subunit A (PR 65), alpha isoform PPP2R1A Hs.467192 AK090488 19q13 <0.05 16.04 <0.05 12.64
    Protein phosphatase 2 (formerly 2A), regulatory subunit B (PR 52), beta isoform PPP2R2B Hs.193825 M64930 5q31-5q32 <0.01* 29.87
    Protein phosphatase 2, regulatory subunit B (B56), gamma isoform PPP2R5C Hs.368264 NM_002719 14q32 <0.01 18.07
    Protein phosphatase 3 (formerly 2B), catalytic subunit, alpha isoform (calcineurin A alpha) PPP3CA Hs.435512 NM_000944 4q21-q24 <0.01* 43.20 <0.01* 26.06
    Protein phosphatase 3 (formerly 2B), catalytic subunit, beta isoform (calcineurin A beta) PPP3CB Hs.500067 BC028049 10q21-q22 <0.01* 37.18 <0.01* 47.07 <0.01 17.08
    Protein phosphatase 3 (formerly 2B), regulatory subunit B, 19kDa, alpha isoform (calcineurin B, type I) PPP3R1 Hs.280604 BC027913 2p15 <0.01* 33.47 <0.01* 31.25
    AP1
    V-fos FBJ murine osteosarcoma viral oncogene homolog FOS Hs.25647 BX647104 14q24 <0.01* -55.69 <0.01* -46.49
    Small G protein
    Rho guanine nucleotide exchange factor (GEF) 3 ARHGEF3 Hs.476402 AL833224 3p21-p13 <0.01* 28.51
    Ras homolog gene family, member I ARHI Hs.194695 AK096393 1p31 <0.05 20.60 <0.01* 28.06
    DIRAS family, GTP-binding RAS-like 2 DIRAS2 Hs.165636 M_017594 9q22 <0.01* 63.78
    RAB31, member RAS oncogene family RAB31 Hs.99528 NM_006868 18p11 <0.01* -27.47
    Rab acceptor 1 (prenylated) RABAC1 Hs.11417 BE779053 19q13 <0.01 16.11
    RAN binding protein 6 RANBP6 Hs.167496 BX537405 9p24 <0.05 17.28
    RAP1, GTPase activating protein 1 RAP1GA1 Hs.148178 BC035030 1p36-p35 <0.01* 22.75
    Hypothetical protein LOC145899 RASGRF1 Hs.459035 NM_002891 15q24 <0.01* 26.84
    RAS guanyl releasing protein 1 (calcium and DAG-regulated) RASGRP1 Hs.511010 AF081195 15q15 <0.05 18.52
    RAS guanyl releasing protein 3 (calcium and DAG-regulated) RASGRP3 Hs.143674 BC027849 2p25-p24 <0.01* -20.95
    Rho-related BTB domain containing 3 RHOBTB3 Hs.445030 NM_014899 5q15 <0.01* -24.67
    Ras and Rab interactor 2 RIN2 Hs.472270 NM_018993 <0.01* -37.60
    Ras-like without CAAX 2 RIT2 Hs.464985 AL713637 18q12 <0.01* 29.34
    Rho family GTPase 1 RND1 Hs.124940 AK124288 12q12-q13 <0.01* 28.02
    V-Ki-ras2 Kirsten rat sarcoma 2 viral oncogene homolog KRAS2 Hs.505033 NM_033360 12p12 <0.01* 20.01
    Ras-GTPase activating protein SH3 domain-binding protein 2 G3BP2 Hs.303676 NM_203505 4q21 <0.01* 25.13 <0.05 17.74
    Potassium channel
    Potassium channel modulatory factor 1 KCMF1 Hs.345694 NM_020122 2p11 <0.01* 23.55
    Potassium voltage-gated channel, shaker-related beta 2 KCNAB2 Hs.440497 AK124696 1p36 <0.05 19.97
    Potassium inwardly-rectifying channel, subfamily J 2 KCNJ2 Hs.1547 NM_000891 17q23-q24 <0.01* -41.14
    Potassium inwardly-rectifying channel, subfamily J 10 KCNJ10 Hs.408960 NM_002241 1q22-q23 <0.01* -25.25
    Potassium channel, subfamily K, member 1 KCNK1 Hs.208544 AL833343 1 q42-q43 <0.05 11.05
    Potassium intermediate/small conductance calcium-activated channel, subfamily N, member 3 KCNN3 Hs.490765 BX649146 1q21 <0.01* -30.93 <0.01* -23.10
    Potassium channel, subfamily V, member 1 KCNV1 Hs.13285 NM_014379 8q22-q24 <0.01* 21.10
    Sodium channel
    Sodium channel, voltage-gated, type II, alpha 2 SCN2A2 Hs.470470 NM_021007 2q23-q24 <0.01* 26.22
    Sodium channel, voltage-gated, type III, beta SCN3B Hs.4865 AB032984 11q24 <0.01* 22.62 <0.01 17.61
    Solute carrier family 12, (potassium-chloride transporter) member 5 SLC12A5 Hs.21413 NM_020708 20q132 <0.01* 26.35 <0.01* 23.76
    Voltage-dependent anion channel 1 VDAC1 Hs.519320 AK122953 5q31 <0.01 18.44
    Calcium Channel
    Calcium channel, voltage-dependent, alpha 2/delta 3 CACNA2D 3 Hs.369421 NM_018398 3p21 <0.01* 31.61
    Calcium channel, voltage-dependent, beta 2 subunit CACNB2 Hs.59093 NM_000724 10p12 <0.01* 32.21
    Calcium channel, voltage-dependent, beta 3 subunit CACNB3 Hs.250712 AK122911 12q13 <0.01* 43.88
    Calcium channel, voltage-dependent, gamma subunit 3 CACNG3 Hs.7235 AK095553 16p12-p13 <0.01* 32.10
    Chloride channel
    Chloride intracellular channel 4 CLIC4 Hs.440544 AL117424 1p36 <0.01* -34.30
    Table 20
    Anterior Cingulate Cortex Dorsalateral Prefrontal Cortex
    Gene Symbol Accession Gene Name Chromosomal Location Unigene clusters Pathway FC BPD FC MDD FC Control T BPD T MDD T Control FC BPD FC MDD FC Control TBPD TMDD T Control
    HSPA2 NM_021979 heat shock 70kDa protein 2 14q24.1 Hs.432648 Chaperone 1.36 0.68 0.69 9.00 -11.58 -12.22 1.21 0.69 1.01 5.95 -12.25 0.36
    SPP1 NM_000582 secreted phosphoprotein 1 (osteopontin, bone sialoprotein I, early T-lymphocyte activation 1) 4q21-q25 Hs.313 Apoptosis 1.28 0.84 0.36 7.67 -5.88 -35.42 1.32 0.86 0.70 8.49 -5.10 -12.35
    TM4SF10 NM_031442 transmembrane 4 superfamily member 10 Xp11.4 Hs.8769 Apoptosis 1.27 0.86 0.75 7.34 -4.73 -10.03 1.48 0.92 0.69 10.52 -2.30 -10.96
    CAT NM_001752 catalase 11p13 Hs.395771 Oxidative Stress 1.22 0.89 0.68 6.28 -3.94 -13.28 1.22 0.85 0.78 6.14 -4.94 -8.30
    S100B NM_006272 S100 calcium binding protein, beta (neural) 21q22.3 Hs.422181 Apoptosis 1.21 0.94 0.68 6.39 -2.25 -14.39 1.19 0.89 0.75 5.16 -3.57 -9.70
    NR4A1 NM_173157 nuclear receptor subfamily 4, group A, member 1 12q13 Hs.1119 Mitochondria 0.83 0.86 1.17 -9.63 -8.30 9.19 0.83 0.90 1.15 8.36 -5.21 7.06
    BZRAP1 NM_004758 benzodiazapine receptor (peripheral) associated protein 1 17q22-q23 Hs.112499 Mitochondria 0.82 1.02 1.16 -7.05 0.68 6.01 1.00 0.96 1.21 0.10 -1.13 5.47
    GSK3B NM_002093 glycogen synthase kinase 3 beta 3q13.3 Hs.282359 Apoptosis 0.76 1.03 1.20 -9.16 0.94 7.11 0.93 1.00 1.05 -2.43 0.02 1.76
    COX7A1 NM_001864 cytochrome c oxidase subunit Vlla polypeptide 1 (muscle) 19q13.1 Hs.421621 Mitochondria 0.73 1.15 1.36 -7.65 3.54 8.57 0.94 1.08 1.16 -1.73 2.08 4.48
    UQCRB NM_006294 ubiquinol-cytochrome c reductase binding protein 8q22 Hs.131255 Mitochondria 1.06 1.20 0.98 1.64 5.37 -0.58 1.15 1.09 1.14 3.48 2.16 3.62
    DUSP1 NM_004417 dual specificity phosphatase 1 5q34 Hs.171695 Oxidative Stress 0.87 0.74 1.10 -4.00 -8.85 2.92 0.84 0.83 1.13 -4.06 -4.54 3.31
    DUSP6 NM_001946 dual specificity phosphatase 6 12q22-q23 Hs.298654 Apoptosis 0.84 0.70 1.59 -4.15 -8.89 12.26 0.96 0.77 1.77 -0.93 -5.46 13.19
    TM4SF10 NM_031442 transmembrane 4 superfamily member 10 Xp11.4 Hs.8769 Apoptosis 1.27 0.86 0.75 7.34 -4.73 -10.03 1.48 0.92 0.69 10.52 -2.30 -10.96
    ATP6VOE NM_003945 ATPase, H+ transporting, lysosomal 9kDa, V0 subunit e 5q35.2 Hs.440165 Lysosyme 1.15 0.88 0.59 2.89 -2.87 -12.33 1.36 0.90 0.63 6.10 -2.23 -10.14
    GLUL S70290 glutamate-ammonia ligase (glutamine synthase) 1q31 Hs.442669 Mitochondria 1.00 0.67 0.78 -0.07 -7.65 -5.22 1.33 0.78 0.70 4.96 -4.42 -7.06 .
    SPP1 NM_000582 secreted phosphoprotein 1 (osteopontin, bone sialoprotein I, early T-lymphocyte activation 1) 4q21-q25 Hs.313 Apoptosis 1.28 0.84 0.36 7.67 -5.88 -35.42 1.32 0.86 0.70 8.49 -5.10 -12.35
    APG-1 NM_014278 heat shock protein (hsp110 family) 4q28 Hs.135554 Chaperone 0.98 1.05 1.06 -0.22 0.64 0.92 1.26 1.19 0.95 2.81 2.20 -0.77
    HSPA5 NM_005347 heat shock 70kDa protein 5 (glucose-regulated protein, 78kDa) 9q33-q34.1 Hs.310769 Chaperone 1.07 0.98 0.94 1.78 -0.42 -1.76 1.25 1.19 1.00 5.09 4.08 0.09
    GATM NM_001482 glycine amidinotransferase (L-arginine:glycine amidinotransferase) 15q15.1 Hs.75335 Mitochondria 1.08 0.80 0.61 2.62 -7.64 -18.24 1.23 0.78 0.79 7.34 -8.90 -9.41
    CAT NM_001752 catalase 11p13 Hs.395771 Oxidative Stress 1.22 0.89 0.68 6.28 -3.94 -13.28 1.22 0.85 0.78 6.14 -4.94 -8.30
    HADHB NM_000183 hydroxyacyl-Coenzyme A dehydroge/3-ketoacyl-Coenzyme A thiolase/enoyl-Coenzyme A hydratase (trifunctional protein), beta subunit 2p23 Hs.269878 Mitochondria 1.11 0.88 0.71 3.71 -5.10 -14.11 1.22 0.87 0.74 6.30 -4.74 -10.47
    CCT3 NM_005998 chaperonin 1q23 Hs.1708 Chaperone 1.15 1.07 0.96 4.98 2.43 -1.67 1.22 1.04 1.03 5.97 1.34 0.96
    containing TCP1, subunit 3 (gamma)
    DAD1 NM_001344 defender against cell death 1 14q11-q12 Hs.82890 Apoptosis 1.13 1.00 0.88 4.36 0.15 -5.31 1.21 1.00 0.93 6.31 0.05 -2.86
    HSPA2 NM_021979 heat shock 70kDa protein 2 14q24.1 Hs.432648 Chaperone 1.36 0.68 0.69 9.00 -11.58 -12.22 1.21 0.69 1.01 5.95 -12.25 0.36
    NR4A1 NM_173157 nuclear receptor subfamily 4, group A, member 1 12q13 Hs.1119 Mitochondria 0.83 0.86 1.17 -9.63 -8.30 9.19 0.83 0.90 1.15 -8.36 -5.21 7.06
    NAPG NM_003826 N-ethylmaleimide-sensitive factor attachment protein, gamma 18p11.21 Hs.370431 Mitochondria 0.94 1.07 1.69 -1.32 1.53 12.83 0.83 1.04 1.83 -3.06. 0.60 10.97
    MAPK1 NM_002745 mitogen-activated protein kinase 1 22q11.21 Hs.324473 Mitochondria 0.85 0.99 1.12 -4.35 -0.35 3.34 0.82 0.96 1.07 -4.77 , -1.10 1.82
    DAD1 NM_001344 defender against cell death 1 14q11-q12 Hs.82890 Apoptosis 1.04 1.04 1.39 0.94 0.91 8.60 0.79 1.07 1.46 -3.99 1.14 7.17
    STIP1 NM_006819 stress-induced-phosphoprotein 1 (Hsp70/Hsp90-organizing protein) 11q13 Hs.257827 Chaperone 1.17 1.16 0.97 4.50 4.51 -0.86 1.06 1.23 1.00 1.49 5.06 0.06
    DUSP1 NM_004417 dual specificity phosphatase 1 5q34 Hs.171695 Oxidative Stress 0.87 0.74 1.10 -4.00 -8.85 2.92 0.84 0.83 1.13 -4.06 -4.54 3.31
    SLC25A13 NM_014251 solute carrier family 25, member 13 (citrin) 7q21.3 Hs.9599 Mitochondria 1.06 0.91 0.97 2.33 -3.94 -1.55 1.03 0.83 1.03 1.11 -6.61 1.10
    PER2 NM_022817 period homolog 2 (Drosophila) 2q37.3 Hs.410692 Cycling 0.90 0.83 1.31 -3.22 -5.79 8.81 0.97 0.80 1.35 -0.76 -5.58 8.02
    SST M_001048 somatostatin 3q28 Hs.12409 Apoptosis 1.16 0.88 3.34 3.92 -3.70 36.03 0.85 0.78 3.71 4.96 -7.89 45.04
    DUSP6 M_001946 dual specificity phosphatase 6 12q22 q23 Hs.298654 Apoptosis 0.84 0.70 1.59 -4.15 -8.89 12.26 0.96 0.77 1.77 -0.93 -5.46 13.19
    USP9Y NM_004654 ubiquitin specific protease 9, Y-linked (fat facets-like, Drosophila) Yq11.2 Hs.371255 26S proteasome 1.05 0.85 1.91 0.89 -3.32 13.69 1.17 0.75 2.20 2.39 -4.51 13.38
    HSPA2 NM_021979 heat shock 70kDa protein 2 14q24.1 Hs.432648 Chaperone 1.36 0.68 0.69 9.00 -11.58 -12.22 1.21 0.69 1.01 5.95 -12.25. 0.36
    SEMA6A NM_020796 sema domain, transmembrane domain (TM), and cytoplasmic domain, (semaphorin) 6A 5q23.1 Hs.443012 Apoptosis 0.84 0.74 0.68 -4.41 -8.25 -11.00 0.92 0.62 1.10 -1.70 -10.66 2.29
    Table 21
    Accession Symbol Name Cytoband Brain Region Microarray fold change (BPD v Control) QPCR fold change (BPD v Control) Microarray fold change (MDD v Control) QPCR fold change (MDD v Control)
    NM_001001935 ATP5A1 ATP synthase, H+transporting, mitochondrial F1 complex, alpha subunit, isoform 1, cardiac muscle 18q12-q21 DLPFC 1.203 1.587* 1.149 1.135
    NM_004047 ATP6VOB ATPase, H+transporting, lysosomal 21kDa, V0 subunit c" 1p32.3 DLPFC 1.183 1.014 1.074 1.483*
    NM_001696 ATP6V1E1 ATPase, H+ transporting, lysosomal 31kDa, V1 subunit E isoform 1 22pter-q11.2 DLPFC 1.138 1.694* 1.081 0.939
    NM_004458 ACSL4 Acyl-CoA synthetase long-chain family member 4 Xq22.3-q23 DLPFC 1.165 1.266* 1.105 1.095
    NM_174855 IDH3B Isocitrate dehydrogenase 3 (NAD+) beta 20p13 DLPFC 1.148 1.007 1.084 1.189*
    NM_133259 LRPPRC Leucine-rich PPR-motif containing 2p21 DLPFC 1.218 1.62* 1.164 1.802*
    NM_021107 MRPS12 Mitochondrial ribosomal protein S12 19q13.1-q13.2 DLPFC 1.091 1.140 1.110 1.276*
    NM_007103 NDUFV1 NADH dehydrogenase (ubiquinone) flavoprotein 1, 51kDa 11q13 DLPFC 1.115 0.989 1.104 1.430*
    NM_173157 NR4A1 Nuclear receptor subfamily 4, group A, member 1 12q13 DLPFC 0.832 0.584* 0.895 0.516*
    NM_000021 PSEN1 Presenilin 1 (Alzheimer disease 3) 14q24.3 DLPFC 1.081 1.306* 0.882 0.825
    NM_001183 ATP6AP1 ATPase, H+ transporting, lysosomal accessory protein 1 Xq28 AnCg 1.134 1.448 1.112 1.385
    NM_021979 HSPA2 Heat shock 70kDa protein 2 14q24.1 AnCg 1.360 2.552* 0.684 0.703
    NM 174855 IDH3B Isocitrate dehydrogenase 3 (NAD+) beta 20p13 AnCg 1.136 0.998 1.099 0.985
    NM_133259 LRPPRC Leucine-rich PPR-motif containing 2p21 AnCg 1.198 1.079 1.08 1.023
    NM_007103 NDUFV1 NADH dehydrogenase (ubiquinone) flavoprotein 1, 51 kDa 11q13 AnCg 1.169 1.480 1.148 1.107
    NM_173157 NR4A1 Nuclear receptor subfamily 4, group A, member 1 12q13 AnCg 0.833 0.599* 0.859 0.484*
    NM_000021 PSEN1 Presenilin 1 (Alzheimer disease 3) 14q24.3 AnCg 1.109 1.460* 0.845 1.089
    AK125435 PSMB1 Proteasome (prosome, macropain) subunit, beta type, 1 6q27 AnCg 1.271 1.615* 1.020 1.162
    NM_002812 PSMD8 Proteasome (prosome, macropain) 26S subunit, non-ATPase, 8 19q13.2 AnCg 1.155 1.358* 1.104 1.231*
    Table 22
    Accession MtDNA Encoded Gene Symbol Brain Region QPCR fold change QPCR fold change Description start and end bp Table 1 Complex Hs.Unigene
    BPD MDD
    AY882398 MtATP6 ATP6 DLPFC ATP synthase F0 subunit 6 0.853 0.876 Homo sapiens isolate 20_U5a1(Tor13) mitochondrion, complete genome. 8528..9208 Mito paper Table 11 (mt) V Hs.4509
    AY882398 MtATP6 ATP6 AnCg ATP synthase F0 subunit 6 0.588* 0.721* Homo sapiens isolate 20_U5a1(Tor13) mitochondrion, complete genome. 8528..9208 Mito paper Table 11 (mt) V Hs.4509
    AY882398 MtCO1 COX1 DLPFC cytochrome c oxidase , subunit I 0.687 0.875 Homo sapiens isolate 20_U5a1 (Tor13) mitochondrion, complete genome. 5905..7446 Mito paper Table 11 (mt) IV Hs.4512
    AY882398 MtCO1 COX1 AnCg cytochrome c oxidase, subunit I 0.527* 0.808 Homo sapiens isolate 20_U5a1(Tor13) mitochondrion, complete genome. 5905..7446 Mito paper Table 11 (mt) IV Hs.4512
    AY882398 MtCO2 COX2 DLPFC cytochrome c oxidase , subunit II 0.836 1.078 Homo sapiens isolate 20_U5a1(Tor13) mitochondrion, complete genome. 7587..8270 Mito paper Table 11 (mt) IV Hs.4513
    AY882398 MtC02 COX2 AnCg cytochrome c oxidase , subunit II 0.758# 0.895 Homo sapiens isolate 20_U5a1(Tor13) mitochondrion, complete genome. 7587..8270 Mito paper Table 11 (mt) IV Hs.4513
    AY882398 MtND1 ND1 DLPFC NADH dehydrogenase, subunit I 0.563# 1.007 Homo sapiens isolate 20_U5a1(Tor13) mitochondrion, complete genome. 3308..4264 Mito paper Table 11 (mt) I Hs.4535
    AY882398 MtND1 ND1 AnCg NADH dehydrogenase, subunit I 0.855 0.957 Homo sapiens isolate 20_U5a1 (Tor13) 3308..4264 Mito paper Table 11 (mt) I Hs.4535
    mitochondrion, complete genome.
    AY882398 MtND2 ND2 DLPFC NADH dehydrogenase, subunit II 0.539 0.921 Homo sapiens isolate 20_U5a1(Tor13) mitochondrion, complete genome. 4471..5514 Mito paper Table 11 (mt) I Hs.4536
    AY882398 MtND3 ND3 DLPFC NADH dehydrogenase, subunit III 1.074 0.856 Homo sapiens isolate 20_U5a1(Tor13) mitochondrion, complete genome. 10060..1040 5 Mito paper Table 11 (mt) I Hs.4537
    AY882398 MtND3 ND3 AnCg NADH dehydrogenase, subunit III 0.752# 0.918 Homo sapiens isolate 20_U5a1(Tor13) mitochondrion, complete genome. 10060..1040 5 Mito paper Table 11 (mt) I Hs.4537
    AY882398 MtATP6 ATP6 DLPFC ATP synthase F0 subunit 6 0.853 0.876 Homo sapiens isolate 20_U5a1(Tor13) mitochondrion, complete genome. 8528..9208 Mito paper Table 11 (mt) V Hs.4509
    * significant at p <0.05 two-tailed
    # trend p < 0.1 two-tailed
    Table 23
    Primer Forward Reverse
    VASE 5'-GACCCCATTCCCTCCATCAC-3' 5'-GGCTACGCACCACCATGTG-3'
    Exon a 5"-GACGCAGCCAGTCCATAGC-3" *1
    Exon b 5'-CGTCTACCCCTGTTCCATTGTC-3' 5'-TCTGGTGGAGACAATGGAACAG-3'
    Exon c 5'-TCCTGCCCTTGCAACCA-3' 5'-GGTTGCAAGGGCAGGAAGA-3'
    SEC exon 5'-CCAAGCTGGTCTTCATAATGCTCTA-3' 5'-TTTGATGCTTGAACACTATGAACATG-3'
    Exon 3 5'-GGCGGCGCTCAATGG-3' *2
    Exon 8 *3 5'-GATCAGGTTCACTTTAATAGAGTTTCCA-3'
    SNP9 for sequencing 5'-CGCAGCCAGTCCGTAAGTAAAG-3' 5'-AAGCTGGACCGGCTACTAGGA-3'
    Table 24
    Tests For Genotypic Association (Risk Allele 1)
    Heterozygous Homozygous Allele Positivity
    A/A<->C/A C/C<->A/A [C/C+C/A]<->A/A
    SNP
    9 Odds Ratio [C.I.] χ2 (p-value) Odds Ratio [C.I.] χ2 (p-value) Odds Ratio [C.I.] χ2 (p-value)
    SZ 0.054 [0.003 - 1.16] 6.6 (0.01) 9.57 [0.47 - 193.92] 3.86 (0.049) 0.084 [0.004 - 1.67] 4.88 (0.027)
    SNP b C/C<->T/C T/T<->C/C [T/T+T/C]<->C/C
    BPD 4.05 [1.16 -14.12] 5.33 (0.02) 2.97 [0.78 - 11.30] 2.65 (0.103) 3.66 [1.08 - 11.30] 4.81 (0.028)
    Table 25
    Haplotype Frequency (Odds Ratio)
    SNP 9 - SNP b Control BPD* SZ#
    C-T 0.2 0.19 (0.95) 0.31 (1.54)
    C - (T/C) 0.31 0.46 (1.48) 0.37 (1.19)
    (C/A) - (T/C) 0.22 0.21 (0.95) 0.11 (0.50)
    p (value)
    *BPD vs Control <0.0001
    #SZ vs Control <0.0001
    SZ vs BPD 0.0003
    Table 26
    Polymorphism n Genotype Counts (Frequency) Allele Counts (Frequency) p (Fisher's exact test)
    SNP 9 C/C C/A A/A C A
    Control
    55 33 (0.60) 22 (0.40) 0 (0) 88 (0.80) 22 (0.20)
    BPD 70 47 (0.67) 23 (0.33) 0 (0) 117 (0.84) 23 (.16) 0.466
    SZ 35 24 (0.69) 8 (0.23) 3 (0.09) 56 (0.80) 14 (0.20) 1
    BPD+SZ 105 71 (0.68) 31 (0.30) 3 (0.02) 173 (0.82) 37 (0.18) 0.602
    SNP b T/T T/C C/C. T C
    Control
    55 16 (0.29) 29 (0.53) 10 (0.18) 61 (0.55) 49 (0.45)
    BPD 70 19 (0.27) 47 (0.67) 4 (0.06) 85 (0.61) 55 (.39) 0.402
    SZ 35 13 (0.37) 19 (0.54) 3 (0.09) 45 (0.64) 25 (0.36) 0.24
    BPD+SZ 105 32 (0.30) 66 (0.63) 7 (0.07) 130 (0.62) 80 (0.38) 0.264
    Table 27
    SNP Genotype Splice Variant BPD vs C MDD vs C
    SNP 9 C/C a-b-c 0.052 ↑
    SNP 6 G/C b-c-SEC 0.05 ↑
    SNP b T/T VASE (-) 0056 ↑
    SNP b T/C c-SEC 0.013 ↓
    SNP b T/C NCAM1 Ct Q-PCR 0.053 ↑
    Table 28.1
    UG Cluster Symbol Gene Name Cytoband fc_AnCg
    Hs.549038 1.919839205
    Hs.547062 Transcribed locus 1.773637669
    Hs.483454 CNN3 Calponin 3, acidic 1p22-p21 1.71410127
    Hs.534365 ZNF43 Zinc finger protein 43 (HTF6) 19p13.1-p12 1.653473114
    Hs.534314 EIF5A Eukaryotic translation initiation factor 5A 17p13-p12 1.616760481
    Hs.427236 Transcribed locus 1.57275662
    Hs.336957 ZNF479 Zinc finger protein 479 7p11.2 1.561114027
    Hs.534385 THOC4 THO complex 4 17q25.3 1.545094715
    Hs.114084 ENPP7 Ectonucleotide pyrophosphatase/phosphodiesterase 7 17q25.3 1.490770143
    Hs.315369 AQP4 Aquaporin 4 18q11.2-q12.1 1.478027231
    Hs.370410 KIAA1145 KIAA1145 protein 12q22 1.435347843
    Hs.75914 RNP24 Coated vesicle membrane protein 12q24.31 1.419831899
    Hs.406708 ILT7 Leukocyte immunoglobulin-like receptor, subfamily A (without TM domain), member 4 19q13.4 1.410439598
    Hs.234249 MAPK8IP1 Mitogen-activated protein kinase 8 interacting protein 1 11p12-p11.2 1.371562585
    Hs.534525 LOC114984 Hypothetical protein BC014089 16p13.3 1.368785293
    Hs.212838 A2M Alpha-2-macroglobulin 12p13.3-p12.3 1.365561844
    Hs.402752 TAF15 TAF15 RNA polymerase II, TATA box binding protein (TBP)-associated factor, 68kDa 17q11.1-q11.2 1.362205401
    Hs.535415 Ig rearranged gamma-chain mRNA, subgroup VH2, V-D-J region 1.355666889
    Hs.467138 Transcribed locus, moderately similar to XP_507997.1 similar to Kv channel interacting protein 2 isoform 4; A-type potassium channel modulatory protein 2; cardiac voltage gated potassium channel modulatory subunit; Kv channel-interacting protein 2 [Pan tr 1.351924341
    Hs.513600 Transcribed locus 1.347512479
    Hs.380218 Transcribed locus 1.327051286
    Hs.458607 NOPE Likely ortholog of mouse neighbor of Punc E11 15q22.31 1.324220424
    Hs.42034 TCP10L T-complex 10 (mouse)-like 21 q22.11 1.322514626
    Hs.521286 RARRES2 Retinoic acid receptor responder (tazarotene induced) 2 7q36.1 1.31025151
    Hs.25422 CDNA FLJ42519 fis, clone BRACE3000787 1.309835897
    Hs.293379 Transcribed locus 1.307793259
    Hs.356766 Similar to RPE-spondin 20q13.13 1.290259345
    Hs.379010 PSCA Prostate stem cell antigen 8q24.2 1.287232595
    Hs.511757 GJB6 Gap junction protein, beta 6 (connexin 30) 13q11-q12.1 1.287226239
    Hs.59106 CGRRF1 Cell growth regulator with ring finger domain 1 14q22.2 1.284552431
    Hs.83916 NDUFA5 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 5, 13kDa 7q32 1.283862892
    Hs.119878 FLJ34389 Hypothetical protein FLJ34389 16q22.3 1.28271805
    Hs.494261 PSAT1 Phosphoserine aminotransferase 1 9q21.2 1.276727208
    Hs.75969 PNRC1 Proline-rich nuclear receptor coactivator 1 6q15 1.273555895
    Hs.467273 CACNG8 Calcium channel, voltage-dependent, gamma subunit 8 19q13.4 1.270119391
    Hs.280805 MGC20579 Hypothetical protein MGC20579 13q34 1.269164507
    Hs.145480 Transcribed locus 1.266189386
    Hs.511454 PLXNA4 Plexin A4 7q32.3 1.263673467
    Hs.80132 SNX15 Sorting nexin 15 11q12 1.26259913
    Hs.514819 AP2B1 Adaptor-related protein complex 2, beta 1 subunit 17q11.2-q12 1.250901951
    Hs.385772 LOC283914 Hypothetical protein LOC283914 16p11.1 1.247941693
    Hs.151536 RAB13 RAB13, member RAS oncogene family 1q21.2 1.247404054
    Hs.548424 Transcribed locus 1.245303451
    Hs.519930 C6orf62 Chromosome 6 open reading frame 62 6p22.2 1.233111115
    Hs.45140 TMEM35 Transmembrane protein 35 Xq22.1 1.232645678
    Hs.505295 MADP-1 MADP-1 protein 12q12 1.226870897
    Hs.513883 PELP1 Proline-, glutamic acid-, leucine-rich protein 1 17p13.2 1.224880728
    Hs.474836 LOC387593 TPTE/TPIP pseudogene 22q13 1.223415544
    Hs.115284 ZNF213 Zinc finger protein 213 16p13.3 1.223309419
    Hs.369624 15E1.2 Hypothetical protein 15E1.2 12q24.31 1.223144605
    Hs.467960 RAB10 RAB10, member RAS oncogene family 2p23.3 1.222273799
    Hs.533282 NONO Non-POU domain containing, octamer-binding Xq13.1 1.219220007
    Hs.181272 PKD2 Polycystic kidney disease 2 (autosomal dominant) 4q21-q23 1.212879872
    Hs.80720 GAB1 GRB2-associated binding protein 1 4q31.21 1.211569918
    Hs.123464 P2RY5 Purinergic receptor P2Y, G-protein coupled, 5 13q14 1.210914564
    Hs.507185 ZFPM1 Zinc finger protein, multitype 1 16q24.2 1.210160133
    Hs.5324 C2orf25 Chromosome 2 open reading frame 25 2q23.3 1.209212561
    Hs.374847 LOC400794 Hypothetical gene supported by BC030596 1q23.2 1.208951568
    Hs.517792 C3orf10 Chromosome 3 open reading frame 10 3p25.3 1.208760837
    Hs.517352 PRODH Proline dehydrogenase (oxidase) 1 22q11.21 1.206253745
    Hs.483561 ORF1-FL49 Putative nuclear protein ORF1-FL49 5q31.2 1.205748855
    Hs.75640 NPPA Natriuretic peptide precursor A 1 p36.21 1.20542617
    Hs.491695 UBE2V2 Ubiquitin-conjugating enzyme E2 variant 2 8q11.21 1.204850064
    Hs.335057 NEDD5 Neural precursor cell expressed, developmentally down-regulated 5 2q37 1.202774491
    Hs.28280 SLC35F4 Solute carrier family 35, member F4 14q22.2 1.201026204
    Table 28.2
    UG Cluster Symbol Gene Name Cytoband fc_AnCg
    Hs.134974 GAP43 Growth associated protein 43 3q13.1-q13.2 0.833001805
    Hs.444637 LRP8 Low density lipoprotein receptor-related protein 8, apolipoprotein e receptor 1p34 0.832591468
    Hs.268849 GLO1 Glyoxalase I 6p21.3-p21.1 0.832479968
    Hs.435952 CDK5RAP1 CDK5 regulatory subunit associated protein 1 20pter-q11.23 0.831820461
    Hs.360940 dJ222E13.1 Kraken-like 22q13 0.828943053
    Hs.500721 MMS19L MMS19-like (MET18 homolog, S. cerevisiae) 10q24-q25 0.827060597
    Hs.200285 TCF4 Transcription factor 4 18q21.1 0.826908526
    Hs.2890 PRKCG Protein kinase C, gamma 19q13.4 0.82630472
    Hs.153661 Transcribed locus 0.825857073
    Hs.483924 MRPL22 Mitochondrial ribosomal protein L22 5q33.1-q33.3 0.825300862
    Hs.210385 HERC1 Hect (homologous to the E6-AP (UBE3A) carboxyl terminus) domain and RCC1 (CHC1)-like domain (RLD) 1 15q22 0.82523312
    Hs.353454 FLJ10276 Hypothetical protein FLJ10276 1p35.1 0.825093453
    Hs.157234 MRNA; cDNA DKFZp547A0515 (from clone DKFZp547A0515) 0.824586185
    Hs.75667 SYP Synaptophysin Xp11.23-p11.22 0.824282156
    Hs.451353 Homo sapiens, clone IMAGE:5288537, mRNA 0.823552515
    Hs.514373 MTMR4 Myotubularin related protein 4 17q22-q23 0.823152304
    Hs.102696 MCTS1 Malignant T cell amplified sequence 1 Xq22-q24 0.822759185
    Hs.445503 SYN2 Synapsin II 3p25 0.822588683
    Hs.524094 PS1D Putative S1 RNA binding domain protein 1p35.2 0.822570466
    Hs.522668 UBQLN2 Ubiquilin 2 Xp11.23-p11.1 0.821156426
    Hs.517148 TH1L TH1-like (Drosophila) 20q13 0.821107397
    Hs.380334 ZNF148 Zinc finger protein 148 (pHZ-52) 3q21 0.821027253
    Hs.534575 MGC2198 Hypothetical protein MGC2198 5q35.2 0.819426558
    Hs.47546 C6orf70 Chromosome 6 open reading frame 70 6q27 0.819049461
    Hs.25601 CHD3 Chromodomain helicase DNA binding protein 3 17p13.1 0.818740301
    Hs.460978 APPBP1 Amyloid beta precursor protein binding protein 1, 59kDa 16q22 0.818504577
    Hs.448851 USP6 Ubiquitin specific protease 6 (Tre-2 oncogene) 17q11 0.818420142
    Hs.532755 GTL3 Likely ortholog of mouse gene trap locus 3 16q21 0.816593983
    Hs.98510 WDR44 WD repeat domain 44 Xq24 0.815070227
    Hs.189119 CXXC5 CXXC finger 5 5q31.2 0.814065874
    Hs.134060 FNBP1L Formin binding protein 1-like 1p22.1 0.813237448
    Hs.549821 Data not found 0.812556603
    Hs.78944 RGS2 Regulator of G-protein signalling 2, 24kDa 1 q31 0.811942329
    Hs.535060 LOC441385 9p24.1 0.811837917
    Hs.549166 Data not found 0.811572937
    Hs.224418 Transcribed locus 0.811556308
    Hs.5258 MAGED1 Melanoma antigen, family D, 1 Xp11.23 0.811035568
    Hs.537449 Transcribed focus 0.810141577
    Hs.515545 TBC1D17 TBC1 domain family, member 17 19q13.33 0.809453648
    Hs.268122 LOC51321 Hypothetical protein LOC51321 17q24.2 0.808975121
    Hs.502910 NKIRAS2 NFKB inhibitor interacting Ras-like 2 17q21.2 0.808919755
    Hs.471876 ING5 Inhibitor of growth family, member 5 2q37.3 0.806159689
    Hs.198612 GPR51 G protein-coupled receptor 51 9q22.1-q22.3 0.806097527
    Hs.401509 RBM10 RNA binding motif protein 10 Xp11.23 0.806076882
    Hs.380857 TD-60 RCC1-like 1p36.13 0.805526827
    Hs.49582 PPP1R12A Protein phosphatase 1, regulatory (inhibitor) subunit 12A 12q15-q21 0.805427552
    Hs.515162 CALR Calreticulin 19p13.3-p13.2 0.804252658
    Hs.444558 KHDRBS3 KH domain containing, RNA binding, signal transduction associated 3 8q24.2 0.803823019
    Hs.317632 CDH18 Cadherin 18, type 2 5p15.2-p15.1 0.803613164
    Hs.471104 NOP5/NOP58 Nucleolar protein NOP5/NOP58 2q33.1 0.802352232
    Hs.348526 LOC474358 Hypothetical BC042079 locus 10q23-q25 0.802291667
    Hs.475018 TCF20 Transcription factor 20 (AR1) 22q13.3 0.801897074
    Hs.463074 ATP6VOA1 ATPase, H+ transporting, lysosomal V0 subunit a isoform 1 17q21 0.801766875
    Hs.28144 FSD1 Fibronectin type 3 and SPRY domain containing 1 19p13.3 0.800329999
    Hs.32309 INPP1 Inositol polyphosphate-1-phosphatase 2q32 0.796446965
    Hs.512973 HSPC121 Butyrate-induced transcript 1 15q22.2 0.796376497
    Hs.13245 LPPR4 Plasticity related gene 1 1 p21 .3 0.794472742
    Hs.199743 ME3 Malic enzyme 3, NADP(+)-dependent, mitochondrial 11cen-q22.3 0.794075417
    Hs.523755 FLRT1 Fibronectin leucine rich transmembrane protein 1 11q12-q13 0.792664481
    Hs.515785 BLVRB Biliverdin reductase B (flavin reductase (NADPH)) 19q13.1-q13.2 0.792511112
    Hs.437277 MGAT4B Mannosyl (alpha-1,3-)-glycoprotein beta-1,4-N-acetylglucosaminyltransferase, isoenzyme B 5q35 0.790721678
    Hs.387982 CDNA clone IMAGE:5261489, partial cds 0.789879979
    Hs.136164 TSPYL2 TSPY-like 2 Xp11.2 0.788609957
    Hs.332847 CRIM1 Cysteine-rich motor neuron 1 2p21 0.786568887
    Hs.348493 GPRASP2 G protein-coupled receptor associated sorting protein 2 Xq22.1 0.786263129
    Hs.7736 MRPL27 Mitochondrial ribosomal protein L27 17q21.3-q22 0.785613917
    Hs.177275 ANKRD6 Ankyrin repeat domain 6 6q14.2-q16.1 0.783389812
    Hs.124015 HAGHL Hydroxyacylglutathione hydrolase-like 16p13.3 0.782617204
    Hs.79322 QARS Glutaminyl-tRNA synthetase 3p21.3-p21.1 0.781931967
    Hs.158748 SLC35F3 Solute carrier family 35, member F3 1 q42.2 0.780681609
    Hs.158460 CDK5R2 Cyclin-dependent kinase 5, regulatory subunit 2 (p39) 2q35 0.779713574
    Hs.337730 LCMT1 Leucine carboxyl methyltransferase 1 16p12.3-16p12.1 0.777655555
    Hs.282998 RBM9 RNA binding motif protein 9 22q13.1 0.775799713
    Hs.60300 ZNF622 Zinc finger protein 622 5p15.1 0.775473024
    Hs.405590 EIF3S6 Eukaryotic translation initiation factor 3, subunit 6 48kDa 8q22-q23 0.775166314
    Hs.373952 CAMTA2 Calmodulin binding transcription activator 2 17p13.2 0.775135303
    Hs.528187 Hypothetical gene supported by AK096649 2q33.1 0.774877993
    Hs.536326 Transcribed locus 0.774173869
    Hs.532231 COPG2 Coatomer protein complex, subunit gamma 2 7q32 0.773663441
    Hs.114169 LRRTM2 Leucine rich repeat transmembrane neuronal 2 5q31.3 0.773056562
    Hs.496267 IGBP1 Immunoglobulin (CD79A) binding protein 1 Xq13.1-q13.3 0.771399953
    Hs.190722 HSPC142 HSPC142 protein 19p13.11 0.770969106
    Hs.130197 KIAA1889 KIAA1889 protein 8q12.1 0.770498302
    Hs.436446 ARMET Arginine-rich, mutated in early stage tumors 3p21.1 0.769942554
    Hs.381300 MGC57858 Hypothetical protein MGC57858 6p21.31 0.769698647
    Hs.33191 UNC5A Unc-5 homolog A (C. elegans) 5q35.2 0.769489374
    Hs.350065 PLXNA2 Plexin A2 1 q32.2 0.767903428
    Hs.48372 Full length insert cDNA clone YZ87G11 0.766810973
    Hs.3797 RAB26 RAB26, member RAS oncogene family 16p13.3 0.765394204
    Hs.21925 Transcribed locus 0.764561674
    Hs.336588 LOC147670 Hypothetical protein LOC147670 19q13.43 0.763351376
    Hs.78466 PSMD8 Proteasome (prosome, macropain) 26S subunit, non-ATPase, 8 19q13.2 0.76234581
    Hs.121520 AMIGO2 Amphoterin induced gene 2 12q13.11 0.760653807
    Hs.443731 USP8 Ubiquitin specific protease 8 15q21.2 0.75760815
    Hs.171501 USP11 Ubiquitin specific protease 11 Xp11.23 0.755609225
    Hs.187861 THRB Thyroid hormone receptor, beta (erythroblastic leukemia viral (v-erb-a) oncogene homolog 2, avian) 3p24.3 0.755503889
    Hs.272284 SLITRK4 SLIT and NTRK-like family, member 4 Xq27.3 0.755411535
    Hs.509736 HSPCB Heat shock 90kDa protein 1, beta 6p12 0.755057388
    Hs.188594 Transcribed locus 0.755026908
    Hs.497806 MARK1 MAP/microtubule affinity-regulating kinase 1 1q41 0.751792399
    Hs.549761 Data not found 0.749944
    Hs.479867 CENPC1 Centromere protein C 1 4q12-q13.3 0.749713558
    Hs.135736 NGL-1 Netrin-G1 ligand 11p12 0.747993997
    Hs.463466 CA10 Carbonic anhydrase X 17q21 0.746516358
    Hs.143587 Transcribed locus 0.745018326
    Hs.549196 Data not found 0.744120055
    Hs.534913 Hypothetical gene supported by BC019717 16p11.2 0.743499122
    Hs.523550 ZNF364 Zinc finger protein 364 1q21.1 0.742420643
    Hs.90242 Homo sapiens, clone IMAGE:4796172, mRNA 0.741848633
    Hs.475150 KIAA0767 KIAA0767 protein 22q13.31 0.741559246
    Hs.530698 CHD8 Chromodomain helicase DNA binding protein 8 14q11.2 0.738476834
    Hs.55879 ABCC10 ATP-binding cassette, sub-family C (CFTR/MRP), member 10 6p21.1 0.738455293
    Hs.323537 FLJ12953 Hypothetical protein FLJ12953 similar to Mus musculus D3Mm3e 2p13.1 0.737391028
    Hs.301296 CDNA: FLJ23131 fis, clone LNG08502 0.737354551
    Hs.502461 DGKZ Diacylglycerol kinase, zeta 104kDa 11 p11.2 0.734852399
    Hs.471096 ALS2 Amyotrophic lateral sclerosis 2 (juvenile) 2q33.1 0.73175125
    Hs.92732 PDZK4 PDZ domain containing 4 Xq28 0.727016153
    Hs.537841 Transcribed locus 0.726748437
    Hs.7744 NDUFV1 NADH dehydrogenase (ubiquinone) flavoprotein 1, 51 kDa 11q13 0.726496223
    Hs.100890 RPRM Reprimo, TP53 dependant G2 arrest mediator candidate 2q23.3 0.719810824
    Hs.6132 CPNE6 Copine VI (neuronal) 14q11.2 0.718586854
    Hs.412019 C6orf80 Chromosome 6 open reading frame 80 6q23.1-q24.1 0.715749314
    Hs.119594 CIT Citron (rho-interacting, serine/threonine kinase 21) 12q24 0.712998565
    Hs.518460 AP2M1 Adaptor-related protein complex 2, mu 1 subunit 3q28 0.709632072
    Hs.65425 CALB1 Calbindin 1, 28kDa 8q21.3-q22.1 0.70924145
    Hs.479116 SH3TC1 SH3 domain and tetratricopeptide repeats 1 4p16.1 0.704347818
    Hs.173859 FZD7 Frizzled homolog 7 (Drosophila) 2q33 0.697933574
    Hs.363137 ACAT2 Acetyl-Coenzyme A acetyltransferase 2 (acetoacetyl Coenzyme A thiolase) 6q25.3-q26 0.695447549
    Hs.153648 PPFIA4 Protein tyrosine phosphatase, receptor type, f polypeptide (PTPRF), interacting protein (liprin), alpha 4 1 q32.1 0.694615615
    Hs.524071 Transcribed locus, strongly similar to XP_084672.3 similar to CDNA sequence BC021608 [Homo sapiens] 0.691986755
    Hs.506784 LNK Lymphocyte adaptor protein 12q24 0.688023619
    Hs.516617 SATB2 SATB family member 2 2q33 0.681642635
    Hs.23406 KCTD4 Potassium channel tetramerisation domain containing 4 13q14.12 0.676435668
    Hs.220950 FOXO3A Forkhead box 03A 6q21 0.669990152
    Hs.370549 BCL11A B-cell CLUlymphoma 11A (zinc finger protein) 2p16.1 0.654002694
    Hs.483239 ALDH7A1 Aldehyde dehydrogenase 7 family, member A1 5q31 0.651849727
    Hs.445733 GSK3B Glycogen synthase kinase 3 beta 3q13.3 0.647879909
    Hs.268515 MN1 Meningioma (disrupted in balanced translocation) 1 22q11 0.64462122
    Hs.319503 PTCHD1 Patched domain containing 1 Xp22.11 0.644497163
    Hs.106511 PCDH17 Hypothetical protein LOC144997 13q21.1 0.644270897
    Hs.91448 DUSP14 Dual specificity phosphatase 14 17q12 0.639891125
    Hs.448041 FLJ32363 FLJ32363 protein 5p12 0.633100428
    Hs.22584 PDYN Prodynorphin 20pter-p12 0.630525299
    Hs.215839 DLG2 Discs, large homolog 2, chapsyn-110 (Drosophila) 11q21 0.620144715
    Hs.11899 HMGCR 3-hydroxy-3-methylglutaryl-Coenzyme A reductase 5q13.3-q14 0.619486994
    Hs.23539 CDNA FLJ42249 fis, clone TKIDN2007667 0.614085673
    Hs.314436 NEDL2 NEDD4-related E3 ubiquitin ligase NEDL2 2q32.3 0.605505392
    Hs.490294 KIAA1549 KJAA1549 protein 7q34 0.548338996
    Hs.518469 FLJ10560 Hypothetical protein FLJ10560 3q27.3 0.498445319
    Hs.546322 NOL4 Nucleolar protein 4 18q12 0.491050809
    Hs.282177 PIP5K1C Phosphatidylinositol-4-phosphate 5-kinase, type I, gamma 19p13.3 0.490622069
    Hs.2785 KRT17 Keratin 17 17q12-q21 0.474185311
    Hs.536506 Transcribed locus 0.467413109
    Hs.435001 KLF10 Kruppel-like factor 10 8q22.2 0.463636382
    Hs.537539 Transcribed locus 0.358666831
    Table 29
    v-ATPase Human MDD vs Monkey stressed Array Results
    UGRepAcc Name Symbol Human T-test Affy HIP t2hcmdd.affy Human T-test illu HIP tHCMdd.illu Monkey Midlife Stress change
    BQ230447 ATPase, H+ transporting, lysosomal 9kDa, V0 subunit e ATP6VOE -2.8565 -2.2254 -1.07
    AF245517 ATPase, H+ transporting, lysosomal V0 subunit a isoform 4 ATP6VOA4 -1.6935 -1.5208 1.01
    NM_012463 ATPase, H+ transporting, lysosomal V0 subunit a isoform 2 ATP6V0A2 -0.8322 -1.6802 -1.05
    CR607789 ATPase, H+transporting, lysosomal 13kDa, V1 subunit G isoform 1 ATP6V1G1 -0.0535 -0.0440 -1.08
    AK127853 ATPase, H+ transporting, lysosomal 56/58kDa, V1 subunit B, isoform 1 (Renal tubular acidosis with deafness) ATP6V1B1 0.3202 -0.8313 1.02
    BF214530 ATPase, H+ transporting, lysosomal 31 kDa, V1 subunit E isoform 1 ATP6V1E1 1.3703 1.9643 -1.01
    AK024101 ATPase, H+ transporting, lysosomal 34kDa, V1 subunit D ATP6V1D 1.6380 2.6589 1.09
    NM_001690 ATPase, H+ transporting, lysosomal 70kDa, V1 subunit A ATP6V1A 1.7837 2.4222 1.15
    NM_001695 ATPase, H+transporting, lysosomal 42kDa, V1 subunit C, isoform 1 ATP6V1C1 1.7882 1.0692 -1.01
    AK128641 ATPase, H+ transporting, lysosomal 38kDa, V0 subunit d isoform 1 ATP6V0D1 1.8249 2.4330 1.03
    BC053601 ATPase, H+ transporting, lysosomal 21 kDa, V0 subunit c" ATP6VOB 1.8268 -0.2217 1.10
    AK127505 ATPase, H+ transporting, lysosomal 16kDa, V0 subunit c ATP6VOC 2.5856 2.2705 -1.04
    NM_001693 ATPase, H+ transporting, lysosomal 56/58kDa, V1 subunit B, isoform 2 ATP6V1B2 2.6827 2.6120 1.04
    AK125927 ATPase, H+ transporting, lysosomal V0 subunit a isoform 1 ATP6V0A1 3.3021 1.2269 1.04
    Table 30
    Probe Set Name Identifier LocusLink Name All AD H2O Ave All AD CUS Ave CUS T CUS FC All CUS+AD T All CUS+AD FC
    1370043_at NM_031753 79559 activated leukocyte cell adhesion molecule 8.554991091 8.737278 0.305517 1.134681103 -0.337997115 0.87551622
    1375424_at BE107525 64040 aldehyde dehydrogenase family 9, subfamily A1 7.7269596 7.985884 0.253566 1.196585931 -0.275888751 0.815851533
    1370176_at BG378620 171086 amyotrophic lateral sclerosis 2 (juvenile) chromosome region, candidate 3 8.289171999 8.644384 0.549287 1.279173789 -0.422598222 0.816014485
    1370050_at NM_053311 29598 ATPase, Ca++ transporting, plasma membrane 1 10.02156406 10.47301 0.407594 1.367412006 -0.303601287 0.776657575
    1367585_a_at M28647 24211 ATPase, Na+K+ transporting, alpha 1 10.86677482 10.70967 -0.27986 0.896825718 0.365553735 1.117986929
    1398781_at NM_053884 116664 ATPase, vacuolar, 14 kD 9.794708887 9.635878 -0.40588 0.895750971 0.501130634 1.131514226
    1367595_s_at NM_012512 24223 Beta-2-microglobulin 10.16168978 9.88582 -0.59779 0.825952261 0.384629381 1.135862492
    1370074_at NM_057196 117542 brain-specific angiogenesis inhibitor 1-associated protein 2 8.394268852 8.230674 -0.55098 0.892797502 0.467062082 1.118839281
    1369993_at NM_133605 171140 calcium/calmodul in-dependent protein kinase (CaM kinase) II gamma 8.242780231 8.480603 0.338888 1.179211766 -0.292444973 0.860882267
    1389824_at BF404381 25400 calcium/calmodul in-dependent protein kinase II alpha subunit 7.810218827 7.950381 0.416069 1.102029271 -0.470849794 0.896960966
    1398251_a_at NM_021739 24245 calcium/calmodul in-dependent protein kinase II beta subunit 8.228441025 8.843395 0.474736 1.531509499 -0.367083116 0.701179029
    1367462_at U10861 29156 calpain, small subunit 1 9.116789268 8.937121 -0.50886 0.882905842 0.648775676 1.158940404
    1389876_at BE111167 287005 CaM-kinase II inhibitor alpha 8.411415385 8.683458 0.388539 1.207516219 -0.331393094 0.833810939
    1370853_at AA858621 287005 CaM-kinase II inhibitor alpha 9.552520138 9.824481 0.484035 1.207447775 -0.302368972 0.879487359
    1369215_a_at NM_012836 25306 carboxypeptidase D 7.60221365 7.851067 0.409687 1.188262417 -0.318247147 0.865883498
    1389974_at BF555171 116549 casein kinase II, alpha 1 polypeptide 7.070655253 7.33655 0.576709 1.20238131 -0.509595002 0.849661157
    1387436_at NM_022616 64551 CDC10 (cell division cycle 10, S.cerevisiae, homolog) 8.264076026 7.838606 -0.82282 0.744596339 0.558756443 1.170398833
    1370922_at L15011 29145 cortexin 10.52034023 10.29252 -0.39042 0.853925826 0.642216784 1.297259097
    1368059_at NM_053955 117024 crystallin, mu 9.15043777 8.934658 -0.55932 0.861080678 0.40726188 1.109586413
    1370438_at AF037071 192363 C-terminal PDZ domain ligand of neuronal nitric oxide synthase 8.478106643 8.941752 0.528156 1.37902155 -0.286782718 0.819872562
    1370810_at L09752 64033 cyclin D2 7.552338207 7.824805 0.627432 1.207871164 -0.510232497 0.856351578
    1370180_at AA891213 94267 diphosphoinositol polyphosphate phosphohydolase type II 9.720407691 9.551001 -0.32062 0.889208564 0.33907154 1.112564538
    1399090_at AA944459 252902 dynein, cytoplasmic, light intermediate chain 1 8.708970325 9.160194 0.592983 1.36719938 -0.611477208 0.713171499
    1370048_at NM_053936 116744 endothelial differentiation, lysophosphatidic acid G-protein-coupled receptor, 2 8.190874036 8.434857 0.460008 1.184257781 -0.276403406 0.88926266
    1370341_at AF019973 24334 enolase 2, gamma 10.25966095 10.09239 -0.27444 0.890528386 0.511287186 1.226632739
    1367958_at NM_024397 79249 eps8 binding protein (e3B1), alternatively spliced 7.501336434 7.734993 0.390605 1.175811622 -0.214182288 0.899358171
    1373067_at AI102738 ESTs 9.097780684 9.795279 0.560829 1.621690153 -0.394405591 0.691167336
    1375687_at BE097926 ESTs 9.102908418 9.589148 0.67554 1.400788553 -0.668809291 0.710828697
    1375343_at BE116572 ESTs 9.636710979 10.05569 0.637477 1.336979858 -0.600017235 0.721789025
    1390722_at AW531272 ESTs 8.052433351 8.445177 0.563541 1.312887882 -0.564580433 0.737665796
    1371776_at AA819268 ESTs 8.792612117 9.070286 0.439984 1.21223885 -0.60941328 0.751754214
    1377029_at AI235414 ESTs 7.519789299 8.066421 0.538435 1.460671268 -0.347221628 0.758851202
    1374002_at AI045904 ESTs 7.891491864 8.3451 0.66405 1.369460535 -0.489475041 0.773784892
    1372183_at AI230596 ESTs 7.499883481 7.870917 0.59093 1.293279024 -0.538050749 0.787917813
    1390100_s_at BG371810 ESTs 8.847513531 9.335307 0.623353 1.402298615 -0.383584226 0.796768397
    1376463_at AA955579 ESTs 8.580618929 8.884818 0.477979 1.234732891 -0.512299476 0.797964077
    1380433_at AI229240 ESTs 8.198656524 8.544649 0.807649 1.271025354 -0.703481364 0.812645109
    1376911_at BM386385 ESTs 8.998969791 9.304256 0.378751 1.235663347 -0.292496741 0.834798828
    1374276_at BE104102 ESTs 8.042219162 8.352924 0.479598 1.24031378 -0.357883889 0.843306633
    1393268_at AI071071 ESTs 7.760844879 8.008586 0.670383 1.187346647 -0.604731486 0.847734973
    1385889_at AA893212 ESTs 7.456567924 7.681123 0.541819 1.168417012 -0.524932857 0.856389756
    1388985_at AI012869 ESTs 10.3902729 10.71161 0.771777 1.249486875 -0.374963851 0.861337025
    1375144_at BM388843 ESTs 9.305640348 9.77995 0.689094 1.389253394 -0.247358915 0.862096412
    1375850_at BG371810 ESTs 10.32943696 10.5907 0.646492 1.198530654 -0.463027833 0.863301098
    1376685_at AW532489 ESTs 7.03405558 7.31854 0.585335 1.217975253 -0.362402037 0.873963777
    1375538_at AI230737 ESTs 7.60108064 7.77676 0.590257 1.129495994 -0.611495294 0.87898368
    1377232_at BF406608 ESTs 7.50385238 7.649913 0.456061 1.106543988 -0.555511902 0.885897068
    1374485_at Al137762 ESTs 7.635036986 7.803592 0.478724 1.123932187 -0.480827091 0.88597741
    1389104_s_at BF388420 ESTs 7.793627056 7.97877 0.44046 1.136929557 -0.340035773 0.897093342
    1372790_at BG671530 ESTs 9.913991148 9.6554 -0.42594 0.835903569 0.333317197 1.121671745
    1388738_at AI411227 ESTs 9.013127784 8.836253 -0.32841 0.884617394 0.421781411 1.134002323
    1389600_at AW524433 ESTs 9.341707252 9.136027 -0.31744 0.867129737 0.294052169 1.146455757
    1388195_at AW140475 ESTs 8.841214015 8.623431 -0.48629 0.859885815 0.493109601 1.177343855
    1389867_at BI281086 ESTs 9.622618423 9.295352 -0.44041 0.797045168 0.527626149 1.325364171
    1371977_at BG381477 ESTs, Highly similar to actin related protein 2/3 complex, subunit 3 (21 kDa); Arp2/3 complex subunit p21-Arc [Mus musculus] [M.musculus] 8.22534466 7.988656 -0.43238 0.848690922 0.274472639 1.102307933
    1388683_at AI411174 ESTs, Highly similar to hypothetical protein MGC14151 [Homo sapiens] [H.sapiens] 8.66963072 8.511747 -0.43469 0.896338715 0.466659704 1.111224693
    1375245_at AA800669 ESTs, Highly similar to A36180 61 K transforming protein - human [H.sapiens] 10.09883803 9.917245 -0.40076 0.881728933 0.482696461 1.136988127
    1383054_at BE111631 ESTs, Highly similar to I48724 zinc finger protein PZF - mouse [M.musculus] 7.47092638 7.691487 0.684678 1.165186416 -0.438439828 0.895791948
    1389957_at BG378149 ESTs, Highly similar to JW0059 mtprd protein -mouse [M.musculus] 9.604565474 9.852639 0.339593 1.187620315 -0.23684341 0.879305448
    1374593_at AA799421 ESTs, Highly similar to KPCE_RAT PROTEIN KINASE C, EPSILON TYPE (NPKC-EPSILON) [R.norvegicus] 8.436415302 8.757416 0.610494 1.249196498 -0.371471138 0.855707419
    1375119_at BI284798 ESTs, Highly similar to S70642 ubiquitin ligase Nedd4 - rat (fragment) [R.norvegicus] 9.262421398 9.747071 0.469691 1.399245716 -0.269750886 0.795573103
    1375305_at BI282028 ESTs, Highly similar to ST1B_MOUSE Syntaxin 1 B (P35B) [R.norvegicus] 10.64049686 10.86651 0.543331 1.169598117 -0.407433833 0.884962553
    1390423_at BE104245 ESTs, Highly similar to T14792 hypothetical protein DKFZp586G0322. 1 - human (fragment) [H.sapiens] 8.796017809 9.069358 0.231788 1.208602479 -0.333710591 0.783587436
    1398971_at BI283725 ESTs, Moderately similar to KIAA0100 gene product [Homo sapiens] [H.sapiens] 9.374401563 9.553263 0.485723 1.13199048 -0.397357886 0.8957128
    1388850_at BG671521 ESTs, Moderately similar to HS9B_RAT Heat shock protein HSP 90-beta (HSP 84) [R.norvegicus] 9.638454301 9.957086 0.267318 1.247146979 -0.37021886 0.693183088
    1390592_at BM389412 ESTs, Moderately similar to T14273 zinc finger protein 106 - mouse [M.musculus] 7.927245389 8.209323 0.666237 1.215944997 -0.373953853 0.883872321
    1390097_at BI281738 ESTs, Moderately similar to Y193_HUMAN Hypothetical protein KIAA0193 [H.sapiens] 9.3184521 9.600735 0.359884 1.216117474 -0.289309137 0.840945647
    1371590_s_at BM386159 ESTs, Weakly similar to e-Tropomodulin [Rattus norvegicus] [R.norvegicus] 8.508270545 8.290722 -0.40327 0.860025673 0.715808502 1.286471079
    1390048_at BF408990 ESTs, Weakly similar to hypothetical protein, MNCb-4760 [Mus musculus] [M.musculus] 7.825368912 8.242474 0.608206 1.335245393 -0.241815196 0.891226221
    1375231_a_at BI281838 ESTs, Weakly similar to inhibitor of the DvI and Axin complex [Rattus norvegicus] [R.norvegicus] 9.410742003 9.628854 0.530944 1.163210616 -0.387267422 0.888752574
    1399079_at AI101669 ESTs, Weakly similar to SC65 synaptonemal complex protein [Rattus norvegicus] [R.norvegicus] 9.836360668 10.16998 0.396804 1.260170799 -0.239640185 0.859334251
    1388903_at AI179335 ESTs, Weakly similar to t-complex testis expressed 1 [Rattus norvegicus] [R.norvegicus] 8.332638336 8.114142 -0.5078 0.859460826 0.35815011 1.110966168
    1373063_at BI277000 ESTs, Weakly similar to ubiquitin-conjugating enzyme E2N (homologous to yeast UBC13); bendless protein [Rattus norvegicus] [R.norvegicus] 8.697615098 8.502515 -0.43031 0.87351232 0.463990102 1.15709537
    1371337_at BG378939 ESTs, Weakly similar to S13099 cytochrome-c oxidase (EC 1.9.3.1) chain VIIa precursor - rat [R.norvegicus] 9.813332563 9.642615 -0.3672 0.888400916 0.325054592 1.106521553
    1398846_at BE107346 56783 eukaryotic initiation factor 5 (elF-5) 8.361376446 9.008593 0.495006 1.566143804 -0.414497851 0.664667377
    1387383_at NM_031802 83633 G protein-coupled receptor 51 9.961560372 9.722226 -0.41601 0.847135806 0.740340744 1.292462812
    1368401_at M85035 29627 glutamate receptor, ionotropic, 2 9.7450637 10.12056 0.388163 1.297283064 -0.370340093 0.761334957
    1383189_at AW522430 24416 glutamate receptor, metabotropic 3 7.922810149 8.369635 0.603662 1.363036876 -0.282310414 0.857046994
    1387659_at AF245172 83585 guanine deaminase 8.579875817 8.970425 0.419618 1.310892245 -0.310557432 0.801387052
    1375705_at AI103622 24400 Guanine nucleotide-binding protein beta 1 11.052428 11.43942 0.624776 1.307666247 -0.713058008 0.698548881
    1370053_at BE116953 65040 guanylate kinase associated protein 8.181514198 8.416934 0.402124 1.177248901 -0.304521974 0.873239295
    1375532_at AI008792 25587 Inhibitor of DNA binding 2, dominant negative helix-loop-helix protein 8.470555381 9.278552 0.655617 1.750778223 -0.467889277 0.6448088
    1371148_s_at X52017 24503 internexin, alpha 8.348395102 8.742675 0.442247 1.314286384 -0.351712988 0.790245803
    1370865_at BI277627 25179 isocitrate dehydrogenase 3, gamma 9.508280943 9.334029 -0.41328 0.886227042 0.430076542 1.117948633
    1387071_a_at BE107978 29477 microtubule-associated protein tau 10.20988985 11.06574 0.440253 1.809820398 -0.40915131 0.566925482
    1370831_at AY081195 29254 monoglycecide lipase 7.997950072 8.519811 0.691492 1.435806237 -0.370997892 0.797665306
    1370016_at NM_031070 81734 nel-like 2 homolog (chicken) 9.366440831 9.130241 -0.3398 0.84897858 0.629322922 1.313198487
    1369690_at AI547471 60355 N-ethylmaleimide sensitive factor 9.871860703 9.66198 -0.40131 0.864608826 0.356371725 1.111739806
    1368993_at NM_020088 56762 neurestin 7.578732705 8.063511 0.624639 1.399370323 -0.459690254 0.755545133
    1369404_a_at NM_021767 60391 neurexin 1 7.850829037 8.133737 0.400729 1.216644537 -0.47177386 0.798122143
    1370058_at NM_031783 83613 neurofilament, light polypeptide 9.844994746 9.631533 -0.42188 0.862465188 0.366920816 1.10425063
    1370517_at U18772 266777 neuronal 1 pentraxin 9.516316885 9.313932 -0.51492 0.869112366 0.522032631 1.16884432
    1368255_at NM_017354 50864 neurotrimin 8.456459057 8.628735 0.372202 1.126834366 -0.377704419 0.883893225
    1367851_at J04488 25526 Prostaglandin D synthase 11.49214354 11.29404 -0.3933 0.871692914 0.290393494 1.108310809
    1398790_at NM_017039 24672 Protein phosphatase 2 (formerly 2A), catalytic subunit, alpha isoform 9.819878116 9.61136 -0.34567 0.865425423 0.311060317 1.132692891
    1398825_at D01046 79434 RAB11B, member RAS oncogene family 8.467885101 8.303234 -0.51417 0.892144413 0.494808325 1.119497348
    1370087_at NM_031718 65158 RAB2, member RAS oncogene family 8.267117217 8.013845 -0.58443 0.838991442 0.408100105 1.123112511
    1370372_at AF134409 171099 RASD family, member 2 8.98764962 8.822419 -0.40481 0.891785751 0.417570782 1.12716855
    1369816_at NM_013018 25531 Ras-related small GTP binding protein 3A 9.593343909 9.380739 -0.35073 0.862977618 0.546884115 1.248974009
    1369958_at NM_022542 64373 rhoB gene 8.970515013 8.798449 -0.38451 0.887570983 0.423174482 1.123272591
    1375421_a_at AI600019 192256 rotein carrying the RING-H2 sequence motif 9.594165696 10.03902 0.3913 1.361178681 -0.323072636 0.74473796
    1375621_at AI575254 261737 sideroflexin 5 7.407218485 7.669141 0.495738 1.199075642 -0.417215583 0.848997619
    1370224_at BE113920 25125 signal transducer and activator of transcription 3 7.635173326 7.857439 0.709037 1.166564469 -0.459754483 0.890983344
    1388000_at AF021923 84550 solute carrier family 24 (sodium/potassium /calcium exchanger), member 2 7.301200621 7.512385 0.309079 1.157637879 -0.25114173 0.870463115
    1368440_at NM_017216 29484 solute carrier family 3, member 1 9.398846616 9.93355 0.749691 1.448643968 -0.727328652 0.650057604
    1389986_at AI008409 117556 synaptic vesicle glycoprotein 2 b 8.015724584 8.590117 0.656167 1.489050598 -0.357264595 0.775227175
    1369627_at L10362 117556 synaptic vesicle glycoprotein 2 b 8.663172726 8.82176 0.204444 1.116193334 -0.243485248 0.864656842
    1387662_at L38247 64440 synaptotagmin 4 8.677669502 9.157806 0.309263 1.394875371 -0.334811005 0.692224654
    1369879_a_at NM_019381 24822 Testis enhanced gene transcript 8.733602786 8.580479 -0.38722 0.899301003 0.378082787 1.115748654
    1368841_at NM_053369 84382 transcription factor 4 8.639946736 8.85393 0.486185 1.159885847 -0.374241086 0.892103037
    1386999_at BG380730 56011 tyrosine 3-monooxgenase/try ptophan 5 monooxgenase activation protein, beta polypeptide 8.669827204 8.515457 -0.44737 0.898524607 0.533133344 1.130282208
    1398843_at AI411103 58857 vesicle-associated membrane protein, associated protein a 10.04441626 9.889542 -0.34531 0.898210852 0.35733192 1.10078515
    1386909_a_at AF268467 83529 voltage-dependent anion channel 1 8.025008371 7.83859 -0.4422 0.87878463 0.441711122 1.146114211
    The present invention is further defined by the following numbered embodiments:
    1. 1. A method for determining whether a subject has or is predisposed for a mood disorder, the method comprising the steps of: (i) obtaining a biological sample from a subject; (ii) contacting the sample with a reagent that selectively associates with a polynucleotide or polypeptide encoded by a nucleic acid that hybridizes under stringent conditions to a nucleotide sequence of Table 3-6; and (iii) detecting the level of reagent that selectively associates with the sample, thereby determining whether the subject has or is predisposed for a mood disorder.
    2. 2. The method of embodiment 1, wherein the reagent is an antibody.
    3. 3. The method of embodiment 1, wherein the reagent is a nucleic acid.
    4. 4. The method of embodiment 1, wherein the reagent associates with a polynucleotide.
    5. 5. The method of embodiment 1, wherein the regent associates with a polypeptide.
    6. 6. The method of embodiment 1, wherein the biological sample is obtained from amniotic fluid.
    7. 7. The method of embodiment 1, wherein the mood disorder is selected from the group consisting of bipolar disorder and major depression disorder.
    8. 8. The method of embodiment 1, wherein the level of reagent that associates with the sample is higher than a level associated with humans without a mood disorder.
    9. 9. The method of embodiment 1, wherein the level of reagent that associates with the sample is lower than a level associated with humans without a mood disorder.
    10. 10. A method of identifying a compound for treatment or prevention of a mood disorder, the method comprising the steps of: (i) contacting the compound with a polypeptide, the polypeptide encoded by a polynucleotide that hybridizes under stringent conditions to a nucleic acid sequence comprising a nucleotide sequence listed in Table 3-6 or a nucleic acid sequence of the PSPHL gene; and (ii) determining the functional effect of the compound upon the polypeptide, thereby identifying a compound for treatment or prevention of a mood disorder.
    11. 11. The method of embodiment 10, wherein the contacting step is performed in vitro.
    12. 12. The method of embodiment 10, wherein the polypeptide is expressed in a cell and the cell is contacted with the compound.
    13. 13. The method of embodiment 10, the mood disorder is selected from the group consisting of bipolar disorder and major depression disorder.
    14. 14. The method of embodiment 10, further comprising administering the compound to an animal and determining the effect on the animal.
    15. 15. The method of embodiment 14, wherein the determining step comprises testing the animal's mental function.
    16. 16. A method of identifying a compound for treatment of a mood disorder in a subject, the method comprising the steps of: (i) contacting the compound to a cell, the cell comprising a polynucleotide that hybridizes under stringent conditions to a nucleotide sequence listed Tables 3-10 of a nucleotide sequence of the PSPHL gene; and (ii) selecting a compound that modulates expression of the polynucleotide, thereby identifying a compound for treatment of a mood disorder.
    17. 17. The method of embodiment 16, wherein the expression of the polynucleotide is enhanced.
    18. 18. The method of embodiment 16, wherein the expression of the polynucleotide is decreased.
    19. 19. The method of embodiment 16, further comprising administering the compound to an animal and determining the effect on the animal.
    20. 20. The method of embodiment 19, wherein the determining step comprises testing the animal's mental function.
    21. 21. The method of embodiment 16, wherein the mood disorder is selected from the group consisting of bipolar disorder and major depression disorder.
    22. 22. A method of treating a mood disorder in a subject, the method comprising the step of administering to the subject a therapeutically effective amount of a compound identified using the method of embodiment 10 or embodiment 16.
    23. 23. The method of embodiment 22, wherein the mood disorder is selected from the group consisting of bipolar disorder and major depression disorder.
    24. 24. The method of embodiment 22, wherein the compound is a small organic molecule.
    25. 25. A method of treating a mood disorder in a subject, the method comprising the step of administering to the subject a therapeutically effective amount of a polypeptide, the polypeptide encoded by a polynucleotide that hybridizes under stringent conditions to a nucleotide sequence listed in Tables 3-10 or a nucleotide sequence of the PSPHL gene.
    26. 26. The method of embodiment 25, wherein the mood disorder is selected from the group consisting of bipolar disorder and major depression disorder.
    27. 27. A method of treating a mood disorder in a subject, the method comprising the step of administering to the subject a therapeutically effective amount of a nucleic acid, wherein the nucleic acid hybridizes under stringent conditions to a nucleotide sequence listed Table 3-6 or a nucleic acid sequence of the PSPHL gene.
    28. 28. The method of embodiment 27, wherein the mood disorder is selected from the group consisting of bipolar disorder and major depression disorder.
    29. 29. A method for determining whether a subject has or is predisposed for bipolar disorder, the method comprising the steps of: (i) obtaining a biological sample from a subject;
      • (ii) contacting the sample with a reagent that selectively associates with a polynucleotide or polypeptide encoded by the PSPHL gene; and (iii) detecting the level of reagent that selectively associates with the sample, thereby determining whether the subject has or is predisposed for bipolar disorder.
    30. 30. The method of embodiment 29, wherein the reagent is an antibody.
    31. 31. The method of embodiment 29, wherein the reagent is a nucleic acid.
    32. 32. The method of embodiment 29, wherein the reagent associates with a PSPHL polynucleotide.
    33. 33. The method of embodiment 29, wherein the reagent associates with a PSPHL mRNA.
    34. 34. The method of embodiment 29, wherein the reagent associates with a PSPHL gene.
    35. 35. The method of embodiment 29, wherein the regent associates with a PSPHL polypeptide.
    36. 36. The method of embodiment 29, wherein the biological sample is obtained from amniotic fluid.
    37. 37. The method of embodiment 29, wherein the level of reagent that associates with the sample is lower than a level associated with humans without bipolar disorder.
    38. 38. A method for determining whether a subject has or is predisposed for bipolar disorder, the method comprising the steps of: (i) obtaining a biological sample from a subject; (ii) contacting the sample with a PCR primer pair that selectively binds to the PSPHL gene; and (iii) amplifying the PSPHL gene and detecting the amplified gene product in the sample, thereby determining whether the subject has or is predisposed for bipolar disorder.
    39. 39. A method for determining whether a subject has or is predisposed for a mood disorder, the method comprising: (i) contacting the tissue of one or regions of the subject's brain with a detectably labeled molecule that selectively binds to a gene listed in any of Tables 1-30; (ii) visualizing the distribution of the detectably labeled molecule in the brain tissue; and (iii) correlating the distribution of the detectably labeled molecule with the presence of or predisposition for a mood disorder in the subject.
    40. 40. The method of embodiment 39 wherein said one or more regions are selected from the group consisting of the anterior cingulate cortex (AnCg), dorsolateral prefrontal cortex (DLPFC), cerebellar cortex (CB), superior temporal gyrus (STG), parietal cortex (PC), and nucleus accumbens (nAcc).
    41. 41. The method of embodiment 39 wherein said labeled molecule is an antisense RNA molecule.
    42. 42. The method of embodiment 39 wherein said contacting occurs in vivo.
    43. 43. The method of embodiment 39 wherein said mood disorder is major depressive disorder.
    44. 44. The method of embodiment 39 wherein said mood disorder is bipolar disorder.
    45. 45. A method for determining the course of progression or regression of a mood disorder:
      • (a) measuring in a biological sample, at a first time, a dysregulated transcript associated with said mood disorder from Tables 1-30; (b) measuring, at a second time, said dysregulated transcript in a biological sample from the subject; and (c) comparing the first measurement and the second measurement; wherein the comparative measurements determine the course of the mood disorder.
    46. 46. The method of embodiment 45 wherein said mood disorder is major depressive disorder.
    47. 47. The method of embodiment 45 wherein said mood disorder is bipolar disorder.
    48. 48. A method for treating a subject with a mood disorder, wherein said mood disorder was diagnosed according to a method of any of embodiments 1, 16, 29, or 38, comprising administering a therapeutic formulation which increases the activity of an FGF gene product in said subject.
    49. 49. The method of embodiment 48 wherein said formulation comprises a recombinant FGF gene product.
    50. 50. The method of embodiment 49 wherein said FGF gene product is FGF-2.
    51. 51. The method of embodiment 48 wherein said formulation comprises a vector capable of expressing an FGF gene product in targeted cells of said subject.
    52. 52. The method of embodiment 51 wherein said FGF gene product is FGF-2.
    53. 53. The method of embodiment 48 wherein said mood disorder is MDD.
    54. 54. A method for determining whether a subject has or is predisposed for a mood disorder, the method comprising the steps of: (i) obtaining a biological sample from a subject; (ii) contacting the sample with a reagent that selectively associates with a polynucleotide or polypeptide encoded by a nucleic acid that hybridizes under stringent conditions to a nucleotide sequence of Table 10; and (iii) detecting the level of reagent that selectively associates with the sample, thereby determining whether the subject has or is predisposed for a mood disorder.
    55. 55. A method for determining whether a subject has or is predisposed for a mood disorder, the method comprising the steps of: (i) obtaining a biological sample from a subject; (ii) contacting the sample with a reagent that selectively associates with a polynucleotide or polypeptide encoded by a nucleic acid that hybridizes under stringent conditions to a nucleotide sequence of Tables 18-19; and (iii) detecting the level of reagent that selectively associates with the sample, thereby determining whether the subject has or is predisposed for a mood disorder.
    56. 56. A method for determining whether a subject has or is predisposed for a mood disorder, the method comprising the steps of: (i) obtaining a biological sample from a subject; (ii) contacting the sample with a reagent that selectively associates with a polynucleotide or polypeptide encoded by a nucleic acid that hybridizes under stringent conditions to a nucleotide sequence of Table 20; and (iii) detecting the level of reagent that selectively associates with the sample, thereby determining whether the subject has or is predisposed for a mood disorder.
    57. 57. A method for determining whether a subject has or is predisposed for a mood disorder, the method comprising the steps of: (i) obtaining a biological sample from a subject; (ii) contacting the sample with a reagent that selectively associates with a polynucleotide or polypeptide encoded by a nucleic acid that hybridizes under stringent conditions to a nucleotide sequence of Tables 29-30; and (iii) detecting the level of reagent that selectively associates with the sample, thereby determining whether the subject has or is predisposed for a mood disorder.
    58. 58. The method of embodiment 57, wherein said nucleic acid encodes a V-ATPase subunit and wherein said mood disorder is MDD or chronic stress.
    Figure imgb0043
    Figure imgb0044
    Figure imgb0045
    Figure imgb0046
    Figure imgb0047
    Figure imgb0048
    Figure imgb0049

Claims (10)

  1. An antagonist of FGF9, or an antagonist of a polypeptide comprising an amino acid sequence encoded by a gene listed in any one of Tables 3-6, for use in a method of preventing or treating a major depression disorder in a subject.
  2. An antagonist for use according to claim 1, wherein said subject exhibits any of the following symptoms: persistent sadness, anxiousness, or an "empty" mood; feelings of hopelessness or pessimism; feelings of guilt, worthlessness, or helplessness; loss of interest or pleasure in hobbies and activities that were once enjoyed, including sex; decreased energy, fatigue, being "slowed down"; difficulty concentrating, remembering, or making decisions; insomnia, early-morning awakening, or oversleeping; appetite and/or weight loss or overeating and weight gain; thoughts of death or suicide or suicide attempts; restlessness or irritability; or persistent physical symptoms that do not respond to treatment, such as headaches, digestive disorders, and chronic pain.
  3. An antagonist for use according to claim 1 or 2, wherein said subject is a human.
  4. An antagonist for use according to any one of claims 1 to 3, wherein said antagonist binds to FGF9.
  5. An antagonist that binds FGF9 for use according to claim 4, which comprises an antibody or fragment thereof.
  6. An antagonist for use according to any one of the preceding claims, wherein said method comprises administering to the subject a therapeutically effective amount of an antagonist of FGF9.
  7. An antagonist for use according to claim 6, wherein said therapeutically effective amount is from 1 nanogram to 10 milligrams per kilogram of said subject's body weight.
  8. An antagonist for use according to claim 6, wherein said antagonist is administered orally, nasally, topically, intravenously, intraperitoneally, or intrathecally.
  9. An antagonist for use according to claim 6, 7 or 8, wherein said antagonist is administered with a pharmaceutically acceptable carrier.
  10. Use of a therapeutically effective amount of an antagonist of FGF9 for the manufacture of a medicament for a method of preventing or treating a major depression disorder in a subject.
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Inventor name: SCHATZBERG, ALAN F.

Inventor name: THOMPSON, ROBERT C.

Inventor name: VAWTER, MARQUIS P.

Inventor name: STEIN, RICHARD

Inventor name: JONES, EDWARD G.

Inventor name: LYONS, DAVID M.

Inventor name: CHOUDARY, PRABHAKARA V.

Inventor name: EVANS, SIMON J.

Inventor name: BUNNEY, WILLIAM E.

Inventor name: AKIL, HUDA

Inventor name: MYERS, RICHARD M.

Inventor name: TURNER, CORTNEY A.

Inventor name: LI, JUN

Inventor name: WATSON, STANLEY J.

Inventor name: MOLNAR, MARGHERITA

Inventor name: TOMITA, HIROAKI

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